Humanized anti-cd19 antibodies and their use in treatment of oncology, transplantation and autoimmune disease

ABSTRACT

The present invention provides chimeric and humanized versions of anti-CD19 mouse monoclonal antibodies. The invention further relates to pharmaceutical compositions, immunotherapeutic compositions, and methods using therapeutic antibodies that bind to the human CD19 antigen and that may mediate ADCC, CDC, and/or apoptosis for the treatment of B cell diseases and disorders, such as, but not limited to, B cell malignancies, for the treatment and prevention of autoimmune disease, and for the treatment and prevention of graft-versus-host disease (GVHD), humoral rejection, and post-transplantation lymphoproliferative disorder in human transplant recipients.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/509,457 filed on Oct. 8, 2014; said application Ser. No. 14/509,457is a continuation of U.S. application Ser. No. 13/661,138 filed on Oct.26, 2012, now U.S. Pat. No. 8,883,942, issued Oct. 22, 2014; saidapplication Ser. No. 13/661,138 is a continuation of U.S. applicationSer. No. 11/852,106 filed on Sep. 7, 2007, now U.S. Pat. No. 8,323,653,issued Dec. 4, 2012; and claims benefit under 35 U.S.C. § 119(e) of thefollowing U.S. Provisional Application Nos. 60,939,429, filed May 22,2007, 60/915,309, filed May 1, 2007, 60/911,397, filed Apr. 12, 2007,60/866,917, filed Nov. 22, 2006 and 60/842,935, filed Sep. 8, 2006. Eachof the above listed applications is incorporated by reference herein inits entirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled CD19105US3_seqlistingcreated on Aug. 14, 2014 and having a size of 105 kilobytes.

1. INTRODUCTION

The present invention relates to human, humanized, or chimeric anti-CD19antibodies that bind to the human CD19 antigen. The present invention isalso directed to compositions comprising human, humanized, or chimericanti-CD19 antibodies that may mediate one or more of the following:complement-dependent cell-mediated cytotoxicity (CDC), antigen-dependentcell-mediated-cytotoxicity (ADCC), and programmed cell death(apoptosis). The present invention is further directed to compositionscomprising human, humanized, or chimeric anti-CD19 antibodies of theIgG1 and/or IgG3 human isotype, as well as to compositions comprisinghuman, humanized, or chimeric anti-CD19 antibodies of the IgG2 and/orIgG4 human isotype that may mediate human ADCC, CDC, or apoptosis.

The present invention is further directed to methods for the treatmentof B cell disorders or diseases in human subjects, including B cellmalignancies, using the therapeutic human, humanized, or chimericanti-CD19 antibodies that bind to the human CD19 antigen. The presentinvention is directed to methods for the treatment and prevention ofautoimmune disease as well as the treatment and prevention ofgraft-versus-host disease (GVHD), humoral rejection, andpost-transplantation lymphoproliferative disorder in human transplantrecipients using therapeutic human, humanized, or chimeric anti-CD19antibodies that bind to the human CD19 antigen.

2. BACKGROUND

B cells express a wide array of cell surface molecules during theirdifferentiation and proliferation. Examples include the CD10, CD19,CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a,CD79b, CD80, CD81, CD82, CD83, CD84, CD85, and CD86 leukocyte surfacemarkers. These markers have been generally suggested as therapeutictargets for the treatment of B cell disorders or diseases such as B cellmalignancies, autoimmune diseases, and transplant rejection. Antibodiesthat specifically bind them have been developed, and some have beentested as therapeutic agent for the treatment of diseases and disorders.

For example, chimeric or radiolabeled monoclonal antibody (mAb)-basedtherapies directed against the CD20 cell surface molecule specific formature B cells and their malignant counterparts have been shown to be aneffective in vivo treatment for non-Hodgkin's lymphoma (Tedder et al.,Immunol. Today 15:450-454 (1994); Press et al., Hematology:221-240(2001); Kaminski et al., N. Engl. J. Med. 329:459-465 (1993); Weiner,Semin. Oncol. 26:43-51 (1999); Onrust et al., Drugs 58:79-88 (1999);McLaughlin et al., Oncology 12:1763-1769 (1998); Reff et al., Blood83:435-445 (1994); Maloney et al., Blood 90:2188-2195 (1997); Malone etal., J. Clin. Oncol. 15:3266-3274 (1997); Anderson et al., Biochem. Soc.Transac. 25:705-708 (1997)). Anti-CD20 monoclonal antibody therapy hasalso been found to be partially effective in attenuating themanifestations of rheumatoid arthritis, systemic lupus erythematosus,idiopathic thrombocytopenic purpura and hemolytic anemia, as well asother immune-mediated diseases (Silverman et al., Arthritis Rheum.48:1484-1492 (2002); Edwards et al., Rheumatology 40:1-7 (2001); De Vitaet al., Arthritis Rheumatism 46:2029-2033 (2002); Leandro et al., Ann.Rheum. Dis. 61:883-888 (2002); Leandro et al., Arthritis Rheum.46:2673-2677 (2001)). The anti-CD20 (IgG1) antibody, RITUXAN™(rituximab), has successfully been used in the treatment of certaindiseases such as adult immune thrombocytopcnic purpura, rheumatoidarthritis, and autoimmune hemolytic anemia (Cured et al., WO 00/67796).Despite the effectiveness of these therapies, B cell depletion is lesseffective where B cells do not express or express CD20 at low levels,(e.g., on pre-B cells or immature B cells) or have lost CD20 expressionfollowing CD20 immunotherapy (Smith et al., Oncogene 22:7359-7368(2003)).

Murine monoclonal anti-CD19 antibodies have been described in the art,for example, HD37 (IgG1, kappa) (DAKO North America, Inc, Carpinteria,Calif.), BU12 (Callard et al., J. Immunology, 148(10):2983-7 (1992)),4G7 (IgG1) (Meeker et al., Hybridoma, 3(4):305-20 (1984 Winter)), J4.119(Beckman Coulter, Krefeld, Germany), B43 (PharMingen, San Diego,Calif.), SJ25C1 (BD PharMingen, San Diego, Calif.), FMC63 (IgG2a) (Zolaet al., Immunol. Cell. Biol. 69(PT6): 411-22 (1991); Nicholson et al.,Mol. Immunol., 34:1157-1165 (1997); Pietersz et al., Cancer Immunol.Immunotherapy, 41:53-60 (1995)), 89B(B4) (IgG1) (Beckman Coulter, Miami,Fla.; Nadler et al., J. Immunol., 131:244-250 (1983)), and/or HD237(IgG2b) (Fourth International Workshop on Human LeukocyteDifferentiation Antigens, Vienna, Austria, 1989; and Pezzutto et al., J.Immunol., 138(9):2793-2799 (1987)). Anti-CD19 antibodies or conjugatesthereof have also shown therapeutic potential in various animal modelsof B cell disorders and diseases (Falvell et al., Br. J. Hematol.134(2):157-70 (2006); Vallera et al., Clin. Cancer Res. 11(21):7920-8(2005); Yazawa et al., Proc. Natl. Acad. Sci. USA 102(42):15178-83(2005)).

In particular, the use of humanized CD19 antibodies has been describedfor the treatment of B-cell disease such as lymphoma, leukemia, orautoimmune disease (see, Hansen U.S. Patent Application Publication No.US2005/0070693; U.S. Pat. No. 7,109,304).

Despite recent advances in cancer therapy, B cell malignancies, such asthe B cell subtypes of non-Hodgkin's lymphomas, and chronic lymphocyticleukemia, are major contributors of cancer-related deaths. Accordingly,there is a great need for further, improved therapeutic regimens for thetreatment of B cell malignancies.

Both cellular (T cell-mediated) and humoral (antibody, B cell-mediated)immunity are now known to play significant roles in graft rejection.While the importance of T cell-mediated immunity in graft rejection iswell established, the critical role of humoral immunity in acute andchronic rejection has only recently become evident. Consequently, mostof the advances in the treatment and prevention of graft rejection havedeveloped from therapeutic agents that target T cell activation. Thefirst therapeutic monoclonal antibody that was FDA approved for thetreatment of graft rejection was the murine monoclonal antibodyORTHOCLONE-OKT3™ (muromonab-CD3), directed against the CD3 receptor of Tcells. OKT3 has been joined by a number of other anti-lymphocytedirected antibodies, including the monoclonal anti-CD52 CAMPATH™antibodies, CAMPATH™-1G, CAMPATH™-1H (alemtuzumab), and CAMPATH™-1M),and polyclonal anti-thymocyte antibody preparations (referred to asanti-thymocyte globulin, or “ATG,” also called “thymoglobin” or“thymoglobulin”). Other T cell antibodies approved for the prevention oftransplant rejection include the chimeric monoclonal antibody SIMULECT™(basiliximab) and the humanized monoclonal antibody ZENAPAX™(daclizumab), both of which target the high-affinity IL-2 receptor ofactivated T cells.

The importance of humoral immunity in graft rejection was initiallythought to be limited to hyperacute rejection, in which the graftrecipient possesses anti-donor HLA antibodies prior to transplantation,resulting in rapid destruction of the graft in the absence of aneffective therapeutic regimen of antibody suppression. Recently, it hasbecome evident that humoral immunity is also an important factormediating both acute and chronic rejection. For example, clinicalobservations demonstrated that graft survival in patients capable ofdeveloping class I or class IT anti-HLA alloantibodies (also referred toas “anti-MHC alloantibodies”) was reduced compared to graft survival inpatients that could not develop such antibodies. Clinical andexperimental data also indicate that other donor-specific alloantibodiesand autoantibodies are critical mediators of rejection. For a currentreview of the evidence supporting a role for donor-specific antibodiesin allograft rejection, see Rifle et al., Transplantation, 79:S14-S18(2005). Thus, due to the relatively recent appreciation of the role ofhumoral immunity in acute and chronic graft rejection, currenttherapeutic agents and strategies for targeting humoral immunity areless well developed than those for targeting cellular immunity.Accordingly, there is a need in the art for improved reagents andmethods for treating and preventing graft rejection, i.e.graft-versus-host disease (GVHD), humoral rejection, andpost-transplantation lymphoproliferative disorder in human transplantrecipients.

Autoimmune diseases as a whole cause significant morbidity anddisability. Based on incidence data collected from 1965 to 1995, it hasbeen estimated that approximately 1.2 million persons will develop a newautoimmune disease over the next five years. Jacobsen et al. (ClinImmunol. Immunopathol. 84:223 (1997)) evaluated over 130 publishedstudies and estimated that in 1996, 8.5 million people in the UnitedStates (3.2% of the population) had at least one of the 24 autoimmuncdiseases examined in these studies. Considering the major impact ofautoimmune diseases on public health, effective and safe treatments areneeded to address the burden of these disorders. Thus, there is a needin the art for improved reagents and methods for treating autoimmunedisease.

3. SUMMARY

The present invention relates to human, humanized, or chimeric anti-CD19antibodies that bind to the human CD19 antigen, as well as tocompositions comprising those antibodies. In one embodiment, the presentinvention provides chimeric and humanized versions of anti-CD19 mousemonoclonal antibodies, HB12A and HB12B.

In another embodiment, anti-CD19 antibodies of the invention compriseone, two, three, four, five, or all six of the CDRs of HB12A (cloneB410F12-2-A6-C2 was deposited with the American Type Culture Collection(“ATCC”) on Feb. 11, 2005, ATCC Patent Deposit Designation: PTA-6580) orHB12B (clone B43H12-3-B2-B6 was deposited with the American Type CultureCollection (“ATCC”) on Feb. 11, 2005, ATCC Patent Deposit Designation:PTA-6581).

The amino acid sequences for CDR1, CDR2, and CDR3 of the heavy chainvariable region of HB12A defined according to Kabat are identified asSEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10, respectively. The amino acidsequences for CDR1, CDR2 and CDR3 of the light chain variable region ofHB12A defined according to Kabat are identified as SEQ ID NO:12, SEQ IDNO:14, and SEQ ID NO:16, respectively.

The amino acid sequences for CDR1, CDR2, and CDR3 of the heavy chainvariable region of HB12B defined according to Kabat are identified asSEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26, respectively. The aminoacid sequences for CDR1, CDR2 and CDR3 of the light chain variableregion of HB12B defined according to Kabat are identified as SEQ IDNO:28, SEQ ID NO:30, and SEQ TD NO:32, respectively.

In one embodiment, an anti-CD19 antibody of the invention comprises one,two, three, four, five, or six CDRs having the amino acid sequence of aCDR listed in Table 1, infra.

TABLE 1Residues that are different from the amino acid sequence of the corresponding HB12B parentalCDR appear in bold, underlined. Amino acid residues corresponding to a given variableposition within the consensus CDR sequences (SEQ ID NO.: 230-235) are listed in parenthesis.In specific embodiments, a CDR of the invention may comprise any permeation of theindividual amino acid residues corresponding to variable positions within the CDR.Antibody VH VK Name Domain VH CDR1 VH CDR2 VH CDR3 Domain VK CDR1VK CDR2 VK CDR3 HB12A SEQ. ID SYVMH YFNPYNDG GTYYYGSS SEQ. ID KSSQSLLYLVSKLDS VQGTHFPY NO.: 2 (SEQ. ID TDYYEKFK YPFDY NO.: 4 SNGKTYLN (SEQ. IDT NO.: 6) G (SEQ. ID (SEQ. ID NO.: 14) (SEQ. ID (SEQ. ID NO.: 10)NO.: 12) NO.: 16) NO.: 8) HB12B SEQ. ID SSWMN RIYPGDGD SGFITTVL SEQ. IDRASESVDT AASNQGS QQSKEVPF NO.: 18 (SEQ. ID TNYNGKFK DFDY NO.: 20 FGISFMN(SEQ. ID T NO.: 22) G (SEQ. ID (SEQ. ID NO.: 30) (SEQ. ID (SEQ. IDNO.: 26) NO.: 28) NO.: 32) NO.: 24) 3649 SEQ. ID SSWMN RIYPGDGD SGFITTVLSEQ. ID RASESVDT AASNQGS QQSKEVPF NO.: 34 (SEQ. ID TNYNGKFK DF NO.: 68FGISFMN (SEQ. ID T NO.: 22) G (SEQ. ID (SEQ. ID NO.: 30) (SEQ. ID(SEQ. ID NO.: 26) NO.: 28) NO.: 32) NO.: 24) 7E12 SEQ. ID S T WMNRIYPGDGD SGFITTV Y D SEQ. ID RASESVDT E ASNQGS QQ T KEVPF NO.: 102(SEQ. ID TNYNGKFK F NO.: 110 FGISFIN (SEQ. ID T NO.: 114) G (SEQ. ID(SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 120) NO.: 123) NO.: 126)NO.: 24) 14H5 SEQ. ID SSWMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT EASNQGS QQSKEVPF NO.: 103 (SEQ. ID TNYNGKFK FDY NO.: 111 FGISFMN (SEQ. IDT NO.: 22) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121)NO.: 28) NO.: 32) NO.: 24) 16C9 SEQ. ID SSWMN RIYPGDGD SGFITTV R DSEQ. ID RASESVDT AASNQGS QQSKEVP I NO.: 103 (SEQ. ID TNYNGKFK FDYNO.: 113 FGISFMN (SEQ. ID T NO.: 22) G (SEQ. ID (SEQ. ID NO.: 30)(SEQ. ID (SEQ. ID NO.: 121) NO.: 28) NO.: 127) NO.: 24) 15D1 SEQ. IDSSWMN RIYPGDGD SGFITTV R D SEQ. ID RASESVD H E ASNQGS QQSKEVP I NO.: 104(SEQ. ID TNYN A KFK FDY NO.: 112 FGISFMN (SEQ. ID T NO.: 22) G (SEQ. ID(SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121) NO.: 124) NO.: 127)NO.: 115) 15D7 SEQ. ID SSWMN RIYPGDGD SGFITTV H D SEQ. ID RASESVDT EASNQGS QQSKEVPF NO.: 105 (SEQ. ID TNYNGKFK FDY NO.: 111 FGISFMN (SEQ. IDT NO.: 22) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 122)NO.: 28) NO.: 32) NO.: 24) 16C4 SEQ. ID SSWMN RIYPGDGD SGFITTV R DSEQ. ID RASESVDT E ASNQGS QQSKEVPF NO.: 106 (SEQ. ID TNYN V KFK FDYNO.: 111 FGISFMN (SEQ. ID T NO.: 22) G (SEQ. ID (SEQ. ID NO.: 125)(SEQ. ID (SEQ. ID NO.: 121) NO.: 28) NO.: 32) NO.: 116) 14H5-YG SEQ. IDSSWMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT E ASNQGS QQSKEVPF NO.: 107(SEQ. ID TNY Y GKFK FDY NO.: 111 FGISFMN (SEQ. ID T NO.: 22) G (SEQ. ID(SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121) NO.: 28) NO.: 32)NO.: 117) 14H5-DG SEQ. ID SSWMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT EASNQGS QQSKEVPF NO.: 108 (SEQ. ID TNY D GKGK FDY NO.: 111 FGISFMN(SEQ. ID T NO.: 22) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. IDNO.: 121) NO.: 28) NO.: 32) NO.: 118) 14H5-LG SEQ. ID SSWMN RIYPGDGDSGFITTV R D SEQ. ID RASESVDT E ASNQGS QQSKEVPF NO.: 109 (SEQ. ID TNY LGKFK FDY NO.: 111 FGISFMN (SEQ. ID T NO.: 22) G (SEQ. ID (SEQ. IDNO.: 125) (SEQ. ID (SEQ. ID NO.: 121) NO.: 28) NO.: 32) NO.: 119) 1A7SEQ. ID S V WMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT E ASNQGS QQSKEVPFNO.: 191 (SEQ. ID TNYN V KFK FDY NO.: 111 FGISFMN (SEQ. ID T NO.: 208) G(SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121) NO.: 28)NO.: 32) NO.: 116) 3C3 SEQ. ID S V WMN RIYPGDGD SGFITTV R D SEQ. IDRASESVD H E ASN PY S A QSKEVP I NO.: 191 (SEQ. ID TNYN V KFK FDYNO.: 193 FGISF I N (SEQ. ID T NO.: 208) G (SEQ. ID (SEQ. ID NO.: 218)(SEQ. ID (SEQ. ID NO.: 121) NO.: 211) NO.: 222) NO.: 116) 3E5 SEQ. ID SV WMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT E ASNQGS A QSKEVPF NO.: 191(SEQ. ID TNYN V KFK FDY NO.: 194 FGISFMN (SEQ. ID T NO.: 208) G (SEQ. ID(SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121) NO.: 28) NO.: 223)NO.: 116) 3D4 SEQ. ID S V WMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT EASNQGS A QSK R VPF NO.: 191 (SEQ. ID TNYN V KFK FDY NO.: 195 FGISFMN(SEQ. ID T NO.: 208) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. IDNO.: 121) NO.: 28) NO.: 224) NO.: 116) 3F11 SEQ. ID S V WMN RIY L GDGDSGFITTV R D SEQ. ID RASESV I T E ASNQGS QQSKEVPF NO.: 192 (SEQ. ID TNYNV KFK FDY NO.: 196 FGISFMN (SEQ. ID T NO.: 208) G (SEQ. ID (SEQ. IDNO.: 125) (SEQ. ID (SEQ. ID NO.: 121) NO.: 212) NO.: 32) NO.: 210) 5B5SEQ. ID S V WMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT E ASNQGS A Q T K RVPF NO.: 191 (SEQ. ID TNYN V KFK FDY NO.: 197 FGISFMN (SEQ. ID TNO.: 208) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121)NO.: 28) NO.: 225) NO.: 116) 6F7 SEQ. ID S V WMN RIYPGDGD SGFITTV R DSEQ. ID RASESVDT E ASNQGS QQSKEVP I NO.: 191 (SEQ. ID TNYN V KFK FDYNO.: 198 FGISFMN (SEQ. ID T NO.: 208) G (SEQ. ID (SEQ. ID NO.: 125)(SEQ. ID (SEQ. ID NO.: 121) NO.: 28) NO.: 226) NO.: 116) 1C11 SEQ. IDSSWMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT E ASN PY S QQSKEVPF NO.: 106(SEQ. ID TNYN V KFK FDY NO.: 199 FGISF I N (SEQ. ID T NO.: 22) G(SEQ. ID (SEQ. ID NO.: 218) (SEQ. ID (SEQ. ID NO.: 121) NO.: 213)NO.: 32) NO.: 116) 2B11 SEQ. ID SSWMN RIYPGDGD SGFITTV R D SEQ. IDRASESVDT E ASNQGS A Q T KEVPF NO.: 106 (SEQ. ID TNYN V KFK FDY NO.: 200FGISFMN (SEQ. ID T NO.: 22) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID(SEQ. ID NO.: 121) NO.: 28) NO.: 227) NO.: 116) 2D10 SEQ. ID S V WMNRIYPGDGD SGFITTV R D SEQ. ID RASESVDT E ASNQGS A Q T KEVP N NO.: 191(SEQ. ID TNYN V KFK FDY NO.: 201 FGISFMN (SEQ. ID T NO.: 208) G (SEQ. ID(SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121) NO.: 28) NO.: 228)NO.: 116) 3B4 SEQ. ID S T WMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT EASNQGS QQSKEVPF NO.: 236 (SEQ. ID TNYN V KFK FDY NO.: 111 FGISFMN(SEQ. ID T NO.: 209) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. IDNO.: 121) NO.: 28) NO.: 32) NO.: 116) 5C11 SEQ. ID S V WMN RIYPGDGDSGFITTV R D SEQ. ID RASESV I T E ASN TY S A QSK R VPF NO.: 191 (SEQ. IDTNYN V KFK FDY NO.: 202 FGISFMN (SEQ. ID T NO.: 208) G (SEQ. ID (SEQ. IDNO.: 219) (SEQ. ID (SEQ. ID NO.: 121) NO.: 212) NO.: 224) NO.: 116) 5D4SEQ. ID SSWMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT E ASNQGS QQSKEVPFNO.: 106 (SEQ. ID TNYN V KFK FDY NO.: 203 FGISF R N (SEQ. ID T NO.: 22)G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121) NO.: 214)NO.: 32) NO.: 116) 6C2 SEQ. ID SSWMN RIYPGDGD SGFITTV R D SEQ. IDRASESVDT E ASNQGS QQSKEVP I NO.: 196 (SEQ. ID TNYN V KFK FDY NO.: 198FGISFMN (SEQ. ID T NO.: 22) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID(SEQ. ID NO.: 121) NO.: 28) NO.: 226) NO.: 116) 6C11 SEQ. ID S V WMN RIYL GDGD SGFITIV R D SEQ. ID RASESVDT E ASN P GS QQ T K R VPF NO.: 192(SEQ. ID TNYN V KFK FDY NO.: 204 FGISFMN (SEQ. ID T NO.: 208) G (SEQ. ID(SEQ. ID NO.: 220) (SEQ. ID (SEQ. ID NO.: 121) NO.: 28) NO.: 229)NO.: 210) 9G7 SEQ. ID S V WMN RIYPGDGD SGFITTV R D SEQ. ID RASESV IH EASN R GS A QSKEVP I NO.: 191 (SEQ. ID TNYN V KFK FDY NO.: 205 F GISFMN(SEQ. ID T NO.: 208) G (SEQ. ID (SEQ. ID NO.: 221) (SEQ. ID (SEQ. IDNO.: 121) NO.: 215) NO.: 222) NO.: 116) 1H4 SEQ. ID S V WMN RIYPGDGDSGFITTV R D SEQ. ID RASESVDT E ASN PY S QQSKEVPF NO.: 191 (SEQ. ID TNYNV KFK FDY NO.: 206 FG L SFMN (SEQ. ID T NO.: 208) G (SEQ. ID (SEQ. IDNO.: 218) (SEQ. ID (SEQ. ID NO.: 121) NO.: 216) NO.: 32) NO.: 116) 3C6SEQ. ID S V WMN RIYPGDGD SGFITTV R D SEQ. ID RASESVDT E ASNQGS A Q T K RVPF NO.: 191 (SEQ. ID TNYN V KFK FDY NO.: 197 FGISFMN (SEQ. ID TNO.: 208) G (SEQ. ID (SEQ. ID NO.: 125) (SEQ. ID (SEQ. ID NO.: 121)NO.: 28) NO.: 225) NO.: 116) 5C4 SEQ. ID S V WMN RIYPGDGD SGFITTV R DSEQ. ID RASESV I T E ASN PY S A Q T K R VPF NO.: 191 (SEQ. ID TNYN V KFKFDY NO.: 207 FGISF I N (SEQ. ID T NO.: 208) G (SEQ. ID (SEQ. IDNO.: 218) (SEQ. ID (SEQ. ID NO.: 121) NO.: 217) NO.: 225) NO.: 116)Consensus SEQ ID S(S/T/V) RIY(P/L) SGFITIV SEQ ID RASESV(L/ (E/A)ASN(Q/A)Q(S/ NO.: 237 WMN GDGDTNY (R/L/V/ NO.: 238 D)(T/H)G (P/Q/T)T)K(E/R) (SEQ. ID (N/Y/D/L) H)DFDY (I/L)SF (Y/G)S VP(F/I/N) NO.: 230)(G/A/V)K (SEQ. ID (I/M/R)N (SEQ. ID T FKG NO.: 232) (SEQ. ID NO.: 234)(SEQ. ID (SEQ. ID NO.: 233) NO.: 235) NO.: 231)

In one embodiment, an anti-CD19 antibody of the invention may compriseone or more framework regions of HB12A or HB12B. In one embodiment, anantibody of the invention may further comprise heavy and/or light chainframework (FW) regions from a human antibody (e.g., from a humangermline antibody sequence such as VH3-72, JH4, Vk A10, or Jk4), whereinsaid human framework regions may comprise one or more mutations in whicha human FW residue is exchanged for the corresponding residue present inthe parental mouse (e.g., HB12A or HB12B) heavy or light chain.

In one embodiment, an anti-CD19 antibody of the invention may compriseone or more CDRs having the amino acid sequence of a CDR listed inTable 1. supra and may further comprise one or more heavy chainframework (FW) regions of the VH region designated HB12B-(3-72/JH4) (SEQID NO:34). In another embodiment, an anti-CD19 antibody of the inventioncomprises one or more CDRs having the amino acid sequence of a CDRlisted in Table 1. supra and further comprises one or more heavy chainframework (FW) regions of the VH region designated HB12B-(3-72/JH4) (SEQID NO:34). In one embodiment, an anti-CD19 antibody of the invention maycomprise one or more CDRs having the amino acid sequence of a CDR listedin Table 1. supra and may further comprise one or more light chainframework (FW) regions of the VK region designated HB12B-(A10-Jk4) (SEQID NO:52). In one embodiment, an anti-CD19 antibody of the inventioncomprises one or more CDRs having the amino acid sequence of a CDRlisted in Table 1. supra and further comprises one or more light chainframework (FW) regions of the VK region designated HB12B-(A10-Jk4) (SEQID NO:52). In another embodiment, an anti-CD19 antibody described hereinmay comprise one or more CDRs having the amino acid sequence of a CDRlisted in Table 1. supra, one or more light chain framework regions ofthe VK region designated HB12B-(A10-Jk4), and one or more heavy chainframework regions of the VH region designated HB12B-(3-72/JH4). In afurther embodiment, an anti-CD19 antibody described herein comprises oneor more CDRs having the amino acid sequence of a CDR listed in Table 1.supra, one or more light chain framework regions of the VK regiondesignated HB12B-(A10-Jk4), and one or more heavy chain frameworkregions of the VH region designated HB12B-(3-72/JH4).

For instance, in one embodiment a humanized anti-CD19 antibody of theinvention may comprise a heavy chain variable region which comprisesfour framework regions, FW1, FW2, FW3, and FW4, wherein FW1 comprisesthe amino acid sequence of SEQ ID NO:36, FW2 comprises the amino acidsequence of SEQ ID NO:38, FW3 comprises the amino acid sequence of SEQID NO:40, and FW4 comprises the amino acid sequence of SEQ ID NO:42. Inone embodiment, a humanized anti-CD19 antibody of the inventioncomprises a heavy chain variable region which comprises four frameworkregions, FW1, FW2, FW3, and FW4, wherein FW1 comprises the amino acidsequence of SEQ ID NO:36, FW2 comprises the amino acid sequence of SEQID NO:38, FW3 comprises the amino acid sequence of SEQ ID NO:40, and FW4comprises the amino acid sequence of SEQ ID NO:42.

In addition, a humanized anti-CD19 monoclonal antibody of the inventionmay comprise a light chain variable region comprising four frameworkregions, FW1, FW2, FW3, and FW4, wherein FW1 comprises the amino acidsequence of SEQ ID NO:54; those in which FW2 comprises an amino acidsequence selected from the group consisting of SEQ ID NO:56, SEQ IDNO:64, and SEQ ID NO:72; those in which FW3 comprises an amino acidsequence selected from the group consisting of SEQ ID NO:58, and SEQ IDNO:66; and those in which FW4 comprises the amino acid sequence of SEQID NO:60. In one embodiment, a humanized anti-CDI9 monoclonal antibodyof the invention comprises a light chain variable region comprising fourframework regions, FW1, FW2, FW3, and FW4, wherein FW1 comprises theamino acid sequence of SEQ ID NO:54; those in which FW2 comprises anamino acid sequence selected from the group consisting of SEQ ID NO:56,SEQ ID NO:64, and SEQ ID NO:72; those in which FW3 comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:58, andSEQ ID NO:66; and those in which FW4 comprises the amino acid sequenceof SEQ ID NO:60.

In one embodiment, an anti-CD19 antibody of the invention may comprise aVH comprising the amino acid sequence of SEQ ID NO.:237 or a VLcomprising the amino acid sequence of SEQ ID NO.:238, wherein saidantibody binds a human CD19 antigen. In another embodiment, an anti-CD19antibody of the invention comprises a VH comprising the amino acidsequence of SEQ ID NO.:237 and a VL comprising the amino acid sequenceof SEQ ID NO.:238.

In particular embodiments, an anti-CD19 antibody of the invention maycomprise a light chain variable region selected from the groupconsisting of HB12B VK (SEQ ID NO:20), HB12B-(A10-Jk4) (SEQ ID NO:52),HB12B-364987 (SEQ ID NO:62), HB12B-3649 (SEQ ID NO:68), HB12B-36 (SEQ IDNO:70), HB12A VK (SEQ ID NO:4), 7E12 VK (SEQ ID NO:110), 14H5 VK (SEQ IDNO:111), 15D1 VK (SEQ ID NO:112), 16C9 VK (SEQ ID NO:113), 3C3 VK (SEQID NO:193), 3E5 VK (SEQ ID NO:194), 3D4 VK (SEQ ID NO:195), 3F1 VK (SEQID NO:196), 5B5 VK (SEQ ID NO:197), 6F7 VK (SEQ ID NO:198), 1C11 VK (SEQID NO:199), 2B11 VK (SEQ ID NO:200), 2D10 VK (SEQ ID NO:201), 5C11 VK(SEQ ID NO:202), 5D4 VK (SEQ ID NO:203), 6C11 VK (SEQ ID NO:204), 9G7 VK(SEQ ID NO:205), 1H4 VK (SEQ ID NO:206), and 5C4 VK (SEQ ID NO:207.

In specific embodiments, the present invention further relates to ananti-CD19 antibody comprising a heavy chain variable region selectedfrom the group consisting of HB12B VH (SEQ ID NO:18), HB12B-(3-72/JH4)(SEQ ID NO:34), HB12A VH (SEQ ID NO:2), 7E12 VH (SEQ ID NO:102), 14H5 VH(SEQ ID NO:103), 15D1 VH (SEQ ID NO:104), 15D7 VH (SEQ ID NO:105), 16C4VH (SEQ ID NO:106), 14H5-YG (SEQ ID NO:107), 14H5-DG (SEQ ID NO:108),14H5-LG (SEQ ID NO:109), 1A7 VH (SEQ ID NO:191), 3C3 VH (SEQ ID NO:191),6C11 VH (SEQ ID NO:191), 9G7 (SEQ ID NO:191), 3B4 VH (SEQ ID NO:236),and 3F11 VH (SEQ ID NO:192).

In a particular embodiment, an anti-CD19 antibody of the inventioncomprises the HB12B-3649 (SEQ ID NO:68) light chain variable region andthe HB12B-(3-72/JH4) (SEQ ID NO:34) heavy chain variable region. A DNAclone comprising the humanized anti-hCD19 VH HB12B-(3-72/JH4) wasdeposited with the American Type Culture Collection (“ATCC”) on Oct. 26,2006. A DNA clone comprising the humanized anti-hCD19 VK HB12B-3649 wasdeposited with the American Type Culture Collection (“ATCC”) on Oct. 26,2006.

In one embodiment, a humanized anti-CD19 antibody of the invention maybind to human CD19 with an affinity comparable to that of the mousemonoclonal antibodies HB12A and/or HB12B, or with an affinity comparableto that of the chHB12B antibody comprising HB12B VH (SEQ ID NO:18) andHB12B VK (SEQ ID NO:20).

The invention further provides polynucleotides comprising a nucleotidesequence encoding a human, humanized, or chimeric anti-CD19 antibody ofthe invention or fragments thereof. The invention also encompassespolynucleotides that hybridize under stringent or lower stringencyhybridization conditions, as defined herein, to polynucleotides thatencode a human, humanized, or chimeric antibody that specifically bindsto human CD19.

Another embodiment of the invention is a vector comprising one or morenucleotide sequences encoding a human, humanized, or chimeric anti-CD19antibody described herein or fragments thereof.

The present invention further relates to an isolated cell comprising avector wherein said vector comprises one or more nucleotide sequencesencoding a human, humanized, or chimeric anti-CD19 antibody of theinvention or fragments thereof.

Chimeric, human, and humanized anti-CD19 monoclonal antibodies describedherein include those of the IgGI, IgG2, IgG3, or IgG4 human isotype.

In one embodiment, a humanized anti-CD19 antibody described hereinmediates antibody-dependent cellular cytotoxicity (ADCC),complement-dependent cell-mediated cytotoxicity (CDC), and/or apoptosis.

In a further embodiment, a humanized anti-CD19 antibody described hereininhibits anti-IgM/CpG stimulated B cell proliferation.

The present invention further relates to pharmaceutical compositionscomprising a chimeric, human, and humanized anti-CD19 antibody.

In still another other aspect, the present invention is directed towarda method of treating a B cell malignancy in a human, comprisingadministering to a human in need thereof a therapeutically-effectiveamount of a chimeric, human, or humanized anti-CD19 monoclonal antibody.

In a further aspect, the present invention relates to a method oftreating an autoimmune disease or disorder in a human, comprisingadministering to a human in need thereof a therapeutically-effectiveamount of a chimeric, human, or humanized anti-CD19 monoclonal antibody.

The present invention further relates to a method of treating orpreventing humoral rejection in a human transplant patient, comprisingadministering to a human in need thereof a therapeutically-effectiveamount of a chimeric, human, or humanized anti-CD19 monoclonal antibody.

3.1. Definitions

As used herein, the terms “antibody” and “antibodies” (immunoglobulins)encompass monoclonal antibodies (including full-length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies, humanantibodies, humanized antibodies, camelised antibodies, chimericantibodies, single-chain Fvs (scFv), single-chain antibodies, singledomain antibodies, domain antibodies, Fab fragments, F(ab′)2 fragments,antibody fragments that exhibit the desired biological activity,disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies(including, e.g., anti-Id antibodies to antibodies of the invention),intrabodies, and epitope-binding fragments of any of the above. Inparticular, antibodies include immunoglobulin molecules andimmunologically active fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen-binding site. Immunoglobulin moleculescan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Native antibodies are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain also has regularly spaced intrachaindisulfide bridges. Each heavy chain has at one end a variable domain(VH) followed by a number of constant domains. Each light chain has avariable domain at one end (VL) and a constant domain at its other end;the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Light chains areclassified as either lambda chains or kappa chains based on the aminoacid sequence of the light chain constant region. The variable domain ofa kappa light chain may also be denoted herein as VK. The term “variableregion” may also be used to describe the variable domain of a heavychain or light chain. Particular amino acid residues are believed toform an interface between the light and heavy chain variable domains.Such antibodies may be derived from any mammal, including, but notlimited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice,etc.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in segments called Complementarity Determining Regions(CDRs) both in the light chain and the heavy chain variable domains. Themore highly conserved portions of the variable domains are called theframework regions (FW). The variable domains of native heavy and lightchains each comprise four FW regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FW regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see, Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are generally not involved directly in antigen binding, but mayinfluence antigen binding affinity and may exhibit various effectorfunctions, such as participation of the antibody in ADCC, CDC, and/orapoptosis.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are associated with its binding toantigen. The hypervariable regions encompass the amino acid residues ofthe “complementarity determining regions” or “CDRs” (e.g., residues24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domainand residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chainvariable domain; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop”(e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, J. Mol. Biol., 196:901-917(1987)). “Framework” or “FW” residues are those variable domain residuesflanking the CDRs. FW residues are present in chimeric, humanized,human, domain antibodies, diabodies, vaccibodies, linear antibodies, andbispecific antibodies.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, monoclonal antibodies areadvantageous in that they can be synthesized by hybridoma cells that areuncontaminated by other immunoglobulin producing cells. Alternativeproduction methods are known to those trained in the art, for example, amonoclonal antibody may be produced by cells stably or transientlytransfected with the heavy and light chain genes encoding the monoclonalantibody.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring engineering of theantibody by any particular method. The term “monoclonal” is used hereinto refer to an antibody that is derived from a clonal population ofcells, including any eukaryotic, prokaryotic, or phage clone, and notthe method by which the antibody was engineered. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohleret al., Nature, 256:495 (1975), or may be made by any recombinant DNAmethod (see, e.g., U.S. Pat. No. 4,816,567), including isolation fromphage antibody libraries using the techniques described in Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991), for example. These methods can be used to producemonoclonal mammalian, chimeric, humanized, human, domain antibodies,diabodies, vaccibodies, linear antibodies, and bispecific antibodies.

The term “chimeric” antibodies includes antibodies in which at least oneportion of the heavy and/or light chain is identical with or homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, and atleast one other portion of the chain(s) is identical with or homologousto corresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). Chimeric antibodies of interest herein include“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a nonhuman primate (e.g., Old World Monkey, suchas baboon, rhesus or cynomolgus monkey) and human constant regionsequences (U.S. Pat. No. 5,693,780).

“Humanized” forms of nonhuman (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from nonhumanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which the native CDR residuesare replaced by residues from the corresponding CDR of a nonhumanspecies (donor antibody) such as mouse, rat, rabbit or nonhuman primatehaving the desired specificity, affinity, and capacity. In someinstances, FW region residues of the human immunoglobulin are replacedby corresponding nonhuman residues. Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. In general, a humanized antibody heavy or lightchain will comprise substantially all of at least one or more variabledomains, in which all or substantially all of the CDRs correspond tothose of a nonhuman immunoglobulin and all or substantially all of theFWs are those of a human immunoglobulin sequence. In certainembodiments, the humanized antibody will comprise at least a portion ofan immunoglobulin constant region (Fe), typically that of a humanimmunoglobulin. For further details, see, Jones et al., Nature,321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992).

A “human antibody” can be an antibody derived from a human or anantibody obtained from a transgenic organism that has been “engineered”to produce specific human antibodies in response to antigenic challengeand can be produced by any method known in the art. In certaintechniques, elements of the human heavy and light chain loci areintroduced into strains of the organism derived from embryonic stem celllines that contain targeted disruptions of the endogenous heavy chainand light chain loci. The transgenic organism can synthesize humanantibodies specific for human antigens, and the organism can be used toproduce human antibody-secreting hybridomas. A human antibody can alsobe an antibody wherein the heavy and light chains are encoded by anucleotide sequence derived from one or more sources of human DNA. Afully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, or in vitroactivated B cells, all of which are known in the art.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which non-specific cytotoxic cells (e.g.,Natural Killer (NK) cells, neutrophils, and macrophages) recognize boundantibody on a target cell and subsequently cause lysis of the targetcell. In one embodiment, such cells are human cells. While not wishingto be limited to any particular mechanism of action, these cytotoxiccells that mediate ADCC generally express Fc receptors (FcRs). Theprimary cells for mediating ADCC, NK cells, express FcγRIII, whereasmonocytes express FcγRI, FcγRII, FcγRIII and/or FcγRIV. FcR expressionon hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev.Immunol., 9:457-92 (1991). To assess ADCC activity of a molecule, an invitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecules of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA),95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to initiate complement activation and lyse a target in thepresence of complement. The complement activation pathway is initiatedby the binding of the first component of the complement system (Clq) toa molecule (e.g., an antibody) complexed with a cognate antigen. Toassess complement activation, a CDC assay, e.g., as described inGazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996), may beperformed.

“Effector cells” are leukocytes which express one or more FcRs andperform effector functions. The cells express at least FcγRI, FCγRII,FcγRII and/or FcγRIV and carry out ADCC effector function. Examples ofhuman leukocytes which mediate ADCC include peripheral blood mononuclearcells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cellsand neutrophils.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one embodiment, the FcR is anative sequence human FcR. Moreover, in certain embodiments, the FcR isone which binds an IgG antibody (a gamma receptor) and includesreceptors of the FcγRI, FcγRII, FcγRII, and FcγRIV subclasses, includingallelic variants and alternatively spliced forms of these receptors.FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB(an “inhibiting receptor”), which have similar amino acid sequences thatdiffer primarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (IT1M) in itscytoplasmic domain. (See, Daëron, Annu. Rev. Immunol., 15:203-234(1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol.,9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol.,24:249 (1994)).

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight, non-covalent orcovalent association. It is in this configuration that the three CDRs ofeach variable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Affinity” of an antibody for an epitope to be used in the treatment(s)described herein is a term well understood in the art and means theextent, or strength, of binding of antibody to epitope. Affinity may bemeasured and/or expressed in a number of ways known in the art,including, but not limited to, equilibrium dissociation constant (KD orKd), apparent equilibrium dissociation constant (KD′ or Kd′), and IC50(amount needed to effect 50% inhibition in a competition assay). It isunderstood that, for purposes of this invention, an affinity is anaverage affinity for a given population of antibodies which bind to anepitope. Values of KD′ reported herein in terms of mg IgG per mL ormg/mL indicate mg Ig per mL of serum, although plasma can be used. Whenantibody affinity is used as a basis for administration of the treatmentmethods described herein, or selection for the treatment methodsdescribed herein, antibody affinity can be measured before and/or duringtreatment, and the values obtained can be used by a clinician inassessing whether a human patient is an appropriate candidate fortreatment.

As used herein, the term “avidity” is a measure of the overall bindingstrength (i.e., both antibody arms) with which an antibody binds anantigen. Antibody avidity can be determined by measuring thedissociation of the antigen-antibody bond in antigen excess using anymeans known in the art, such as, but not limited to, by the modificationof indirect fluorescent antibody as described by Gray et al., J. Virol.Meth., 44:11-24. (1993)

An “epitope” is a term well understood in the art and means any chemicalmoiety that exhibits specific binding to an antibody. An “antigen” is amoiety or molecule that contains an epitope, and, as such, alsospecifically binds to antibody.

A “B cell surface marker” as used herein is an antigen expressed on thesurface of a B cell which can be targeted with an agent which bindsthereto. B cell surface markers include the CD10, CD19, CD20, CD21,CD22, CD23, CD24, CD25, CD37, CD53, CD72, CD73, CD74, CD75, CD77, CD79a,CD79b, CD80, CD81, CD82, CD83, CD84, CD85, and CD86 leukocyte surfacemarkers. A B cell surface marker of particular interest ispreferentially expressed on B cells compared to other non-B cell tissuesof a mammal and may be expressed on both precursor B cells and matureB-lineage cells. In one embodiment, the marker is CD19, which is foundon B cells at various stages of differentiation.

The term “antibody half-life” as used herein means a pharmacokineticproperty of an antibody that is a measure of the mean survival time ofantibody molecules following their administration. Antibody half-lifecan be expressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body or a specificcompartment thereof, for example, as measured in serum or plasma, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody half-life results in an increase in mean residencetime (MRT) in circulation for the antibody administered.

The term “isotype” refers to the classification of an antibody's heavyor light chain constant region. The constant domains of antibodies arenot involved in binding to antigen, but exhibit various effectorfunctions. Depending on the amino acid sequence of the heavy chainconstant region, a given human antibody or immunoglobulin can beassigned to one of five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM. Several of these classes may be further divided intosubclasses (isotypes), e.g., IgG1 (gamma 1), IgG2 (gamma 2), IgG3 (gamma3), and IgG4 (gamma 4), and IgA1 and IgA2. The heavy chain constantregions that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively. The structures andthree-dimensional configurations of different classes of immunoglobulinsare well-known. Of the various human immunoglobulin classes, only humanIgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. HumanIgG1 and IgG3 are known to mediate ADCC in humans. Human light chainconstant regions may be classified into two major classes, kappa andlambda

As used herein, the term “immunogenicity” means that a compound iscapable of provoking an immune response (stimulating production ofspecific antibodies and/or proliferation of specific T cells).

As used herein, the term “antigenicity” means that a compound isrecognized by an antibody or may bind to an antibody and induce animmune response.

By the terms “treat,” “treating” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is an inhibition or delay inthe progression of the condition and/or prevention or delay of the onsetof a disease or illness. Thus, the terms “treat,” “treating” or“treatment of” (or grammatically equivalent terms) refer to bothprophylactic and therapeutic treatment regimes.

As used herein, a “sufficient amount” or “an amount sufficient to”achieve a particular result refers to an amount of an antibody orcomposition of the invention that is effective to produce a desiredeffect, which is optionally a therapeutic effect (i.e., byadministration of a therapeutically effective amount). For example, a“sufficient amount” or “an amount sufficient to” can be an amount thatis effective to deplete B cells.

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Stated in anotherway, a “therapeutically effective” amount is an amount that providessome alleviation, mitigation, and/or decrease in at least one clinicalsymptom. Clinical symptoms associated with the disorders that can betreated by the methods of the invention are well-known to those skilledin the art. Further, those skilled in the art will appreciate that thetherapeutic effects need not be complete or curative, as long as somebenefit is provided to the subject.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B: (A) Amino acid sequence alignment of the HB12B VK (SEQ IDNO:20), HB12B-(A10-Jk4) (SEQ ID NO:52), HB12B-364987 (SEQ ID NO:62),HB12B-3649 (SEQ ID NO:68), and HB12B-36 (SEQ ID NO:70) light chainsvariable regions. Sequence residues are numbered according to Kabat. CDRresidues, defined according to Kabat, are boxed. Vernier, Interchain,and Canonical residues of HB12B VK (SEQ ID NO:20) are highlighted inlight gray. Amino acid substitutions of HB12B-364987 (SEQ ID NO:62)(Y40F, K53H, Y91F), HB12B-3649 (SEQ ID NO:68) (Y40F, K53H), and HB12B-36(SEQ ID NO:70) (Y40F) relative to the grafted antibody HB12B-(A10-Jk4)(SEQ ID NO:52) variable domain are highlighted in dark gray. (B) Aminoacid sequence alignment of the HB12B VH (SEQ ID NO:18), HB12B-(3-72/JH4)(SEQ ID NO:34), and HB12B-9m (SEQ ID NO:44) heavy chain variableregions. Sequence residues are numbered according to Kabat. CDRresidues, defined according to Kabat, are boxed. Vernier, Interchain,and Canonical residues of HB12B VH are highlighted in light gray. Aminoacid substitutions of HB12B-9m (SEQ ID NO:44) (L20I, F27Y, T28A, R38I,V49I, F67A, R71A, L80M, I91Y) relative to the grafted antibodyHB12B-(3-72/JH4) (SEQ ID NO:34) variable domain are highlighted in darkgray.

FIG. 2. Binding profile of humanized anti-CD19 antibody #1, comprisingHB12B-(3-72/JH4) VH (SEQ ID NO:34) and HB12B-364987 VK (SEQ ID NO:62),to recombinant human CD19 expressing 300B4 cells in a cell based ELISAassay. OD450 readings for humanized anti-CD19 antibody #1 are markedwith an open square. Chimeric HB12B antibody comprising HB12B VH (SEQ IDNO:18) and HB12B VK (SEQ ID NO:20) was used as a reference standard(closed circle). A human IgG1 antibody of irrelevant specificity wasincluded in the assay as a negative control (open circle). The bindingprofile of humanized anti-CD19 antibody #1 closely matches that of thechimeric anti-CD19 antibody.

FIG. 3. Binding profile of humanized anti-CD19 antibody #1, #2, and #3to recombinant human CD19 expressing 300B4 cells in a cell based ELISAassay. Humanized anti-CD19 antibody #1 comprises HB12B-(3-72/JH4) VH(SEQ ID NO:34) and HB12B-364987 VK (SEQ ID NO:62). Humanized anti-CD19antibody #2 comprises HB12B-(3-72/JH4) VH (SEQ ID NO:34) and HB12B-3649VK (SEQ ID NO:68). Humanized anti-CD19 antibody #3 comprisesHB12B-(3-72/JH4) VH (SEQ ID NO:34) and HB12B-36 VK (SEQ ID NO:70). Thebinding profile of humanized anti-CD19 antibody #1, #2, and #3 is markedwith open squares, open circles, and closed circles, respectively.Chimeric HB12B antibody comprising HB12B VH (SEQ ID NO:18) and HB12B VK(SEQ ID NO:20) was used as reference standard (closed square). Thebinding profile of humanized anti-CD19 antibody #1 and #2 closelymatches that of the chimeric anti-CD19 antibody. The binding ofhumanized anti-CD19 antibody #3 to recombinant human CD19 expressing300B4 cells is significantly weaker than that of the chimeric HB12Bantibody.

FIG. 4. Coomassie stained SDS/PAGE of purified anti-hCD19 antibodies. 1and 5 micrograms of fucosylated (3649) and afucosylated (3649-aFuc)purified humanized anti-CD19 antibody #2 was analyzed by SDS/PAGE. Thepurified preparations are substantially free from contaminatingproteins.

FIG. 5A and FIG. 5B. (A) Mean fluorescence intensity of immunostainedDaudi cells incubated with different concentrations of humanizedanti-CD19 antibody #2. Daudi cells were incubated with differentconcentrations of fucosylated (3649) or afucosylated (3649 aFuc-1 and3649 aFuc-2) anti-CD19 antibody #2. Cells were subsequently stained withRPE conjugated goat anti-human IgG F(ab)′2 and analyzed on a flowcytometer following standard protocols. Daudi cells incubated with ananti-CD20 antibody were included as positive control. The fucosylatedand afucosylated preparations of humanized anti-CD19 antibody #2 displayoverlapping staining profiles. Mean fluorescence intensity of anti-CD19stained cells is lower than that of anti-CD20 stained cells at allantibody concentrations tested. (B) In vitro ADCC activity of humanizedanti-CD19 antibodies. In vitro ADCC activity of fucosylated (3649) andafucosylated (3649-aFuc1 and 3649-aFuc2) preparations of humanizedanti-CD19 antibody #2 was assayed using the CytoTox 96™ kit (Promega)following the manufacturer's instructions. Daudi cells were used astargets. The assay was also performed using a positive control anti-CD20antibody. Both afucosylated preparations of humanized anti-CD19 antibody#2, as well as the positive control anti-CD20 antibody displayedsimilar, robust ADCC activity. The ADCC activity of the fucosylatedanti-CD19 antibody #2 is lower under the conditions used.

FIG. 6. In vitro ADCC activity of humanized anti-CD19 antibodies. Invitro ADCC activity of fucosylated (3649) and afucosylated (3649-aFuc)humanized anti-CD19 antibody #2 was assayed using the CytoTox 96 kit(Promega) following the manufacturer's instructions. Daudi cells servedas targets. An anti-CD20 antibody was used as positive control. An Fcvariant of the anti-CD19 antibody #2 (3649-TM) with abolished ADCC wasused as negative control. The afucosylated humanized anti-CD19 antibody#2 (3649-aFuc) and the positive control anti-CD20 antibody displayedsimilar, robust ADCC activity. The ADCC activity of the fucosylatedhumanized anti-CD19 antibody #2 (3649) is lower under the conditionsused. The negative control Fc variant anti-CD19 antibody #2 showed noADCC activity under the conditions used.

FIG. 7. CD19, CD20, and CD22 expression profile of Raji, Ramos, Daudi,and Namalwa cells. Raji, Ramos, Daudi, and Namalwa cells wereimmunostained with anti-CD19, anti-CD20 or anti-CD22 primary, and PEconjugated goat anti-mouse IgG secondary antibodies and subsequentlyanalyzed on a flow cytometer. The bar graphs represent the ratio of meanchannel fluorescence obtained with immunostained and secondary antibodyonly stained control samples. The RPMI 8226 multiple myeloma cell linethat does not express either CD19, CD20 or CD22 was included as negativecontrol. Significant surface expression of all three molecules isdetected on Raji, Ramos, and Daudi cells. Namalwa cells display CD19 andCD22, but not CD20 on the cell surface.

FIG. 8A/B and FIG. 8C/D. Raji (A), Daudi (B), Ramos (C), and Namalwa (D)cell susceptibility to anti-CD19 #2 mediated ADCC. ADCC assays wereperformed using the CytoTox 96™ kit (Promega) following themanufacturer's instructions. Antibodies used are: (i) the afucosylatedanti-CD19 #2 (3649-aFuc), (ii) the 3M Fc variant of anti-CD19 #2(3649-3M), and (iii) an anti-CD20 control. Effector to target ratio was2.5 to 1. All four cell lines are susceptible to anti-CD19 #2 mediatedADCC. Only Raji, Daudi, and Ramos cells are susceptible to anti-CD20mediated ADCC.

FIG. 9. Fresh tonsillar B cell susceptibility to anti-CD19 #2 mediatedADCC. ADCC assays were performed using the CytoTox 96™ kit (Promega)following the manufacturer's instructions. Antibodies used are: (i) theafucosylated anti-CD19 #2 (3649-aFuc), (ii) the 3M Fc variant ofanti-CD19 #2 (3649-3M), and (iii) an anti-CD20 control. Effector totarget ratio was 2 to 1. Tonsillar B cell are susceptible to ADCCmediated by all three antibodies tested.

FIG. 10A and FIG. 10B. Circulating lymphocytes were isolated form C57B16hCD19 tg+/−, C57B16 hCD19 tg+/+, Balb/c hCD20 tg+/− and Balb/c mice.Isolated cells were stained with PerCP conjugated anti-mouse CD19(α-mCD19), PE conjugated anti-CD3, Alexa488 conjugated anti-human CD19(α-hCD19), and Alexa647 conjugated anti-human CD20 antibodies (α-hCD20).n equals the number of animals analyzed from each group. (A) The meanfluorescence intensity of CD3− cells measured in the hCD19, hCD20, andmCD19 specific channels is presented in a bar graph format. (B)Percentage of CD3− mCD19+ lymphocytes in various genetic backgrounds.

FIG. 11A and FIG. 11B. In vivo B cell depletion by anti-CD19 antibody#2. (A) C57B16 hCD19 tg+/+ and (B) C57B16 hCD19 tg+/− animals weretreated with a single i.v. dose of 250 or 50 μg of anti-CD19 antibody #2(3649). Negative control antibodies used are (i) the ADCC compromised Fcvariant of #2 (3649 TM) and (ii) an antibody of irrelevant specificity(R347). Circulating lymphocytes were isolated 7 days after treatment.Cells were stained with PerCP conjugated anti-mouse CD19 (α-mCD19) andPE conjugated anti-CD3 antibodies. Percentage of mCD19+ CD3− B cells isdisplayed. n equals the number of animals analyzed from each group. Atreatment with a single dose of anti-CD19 antibody #2 resulted in a nearcomplete depletion of B cells.

FIG. 12A and FIG. 12B. In vivo B cell depletion by anti-CD19 antibody#2. C57B16 hCD19 tg+/+ and C57B16 hCD19 tg+/− animals were treated witha single i.v. dose of 250 or 50 μg of anti-CD19 antibody #2 (3649).Negative control antibodies used are (i) the ADCC compromised Fc variantof #2 (3649 TM) and (ii) an antibody of irrelevant specificity (R347).Spleen cells were isolated 7 days after treatment. Cells were stainedwith PerCP conjugated anti-mouse CD19 (α-mCD19) and PE conjugatedanti-CD3 antibodies. Percentage of B cells (mCD19+ CD3−) among spleenlymphocytes is displayed. n equals the number of animals analyzed fromeach group. A treatment with a single dose of anti-CD19 antibody #2resulted in a near complete depletion of B cells.

FIG. 13. Anti-CD19 antibody #2 significantly reduces tumor growth in anin vivo model system. CB17 SCID mice were injected s.c. on the hindflank with 5×106 Raji cells on dayl. Animals were treated with fivebiweekly doses of 10 mg/kg antibody starting on day 4. Antibodies usedare: (i) anti-CD19 #2 (3649), (ii) Fc variant of anti-CD19 #2 withreduced ADCC activity (3649-TM), (iii) anti-CD20, and (iv) isotypecontrol of irrelevant specificity (R347). A group of control animalswere only given PBS. Tumor size was measured twice a week using standardprocedures.

FIG. 14. Anti-CD19 antibody #2 significantly reduces tumor growth in anin vivo model system. CB17 SCID mice were injected s.c. on the hindflank with 5×106 Raji cells on dayl. Animals were treated with fivebiweekly doses of 10 mg/kg or 2.5 mg/kg antibody starting on day 4.Antibodies used are: (i) anti-CD19 #2 at 10 mg/kg or 2.5 mg/kg (3649 10mg/kg and 3649*2.5 mg/kg), (ii) Fc variant of anti-CD19 #2 with reducedADCC activity at 10 mg/kg (3649-TM), (iii) Fc variant of anti-CD19 #2with enhanced human ADCC activity at 10 mg/kg (3649-3M), (iv) anti-CD20at 10 mg/kg, and (v) isotype control of irrelevant specificity at 10mg/kg (R347). A group of control animals were only given PBS. Tumor sizewas measured twice a week using standard procedures.

FIG. 15. Binding profile of 3649, 3649-3M, and 3649-aFuc anti-CD19antibodies to the 158V allele of FcγRIIIA as determined by ELISA. Ananti-CD20 antibody was included in the assay as a reference control. Thebinding affinity of the 3649-3M Fc variant antibody and the 3649-aFucafucosylated antibody to FcγRIIIA is much higher than that of thefucosylated 3649 antibody. The 3649 and anti-CD20 antibodies haveidentical binding profiles.

FIG. 16A/B and FIG. 16C/D/E. FcγRIIIA genotype of effector cellsinfluences the in vitro ADCC activity of the anti-CD19#2 antibody. ADCCassays were performed using the CytoTox 96™ kit (Promega) following themanufacturer's instructions. Antibodies used are: (i) the afucosylatedanti-CD19 #2 (3649-aFuc), (ii) the 3M Fc variant of anti-CD19 #2(3649-3M), and (iii) an anti-CD20 control. Daudi cells served astargets. Either an NK cell line (A) or freshly isolated NK cells (B-E)were used as effector cells. NK cell with V158/V158 (C), V158/F158 (D),and F158/F158 (E) FcγRIIIA genotype were tested. NK cells that compriseat least one copy of the high affinity isoform of FcγRIIIA receptor(V158N158 and V158/F158 genotypes) are more efficient effector cellsthan NK cells homozygous for the low affinity alleles (F158/F158genotype). The observed ADCC activity of the fucosylated antibody (3649)mediated by V158/V158 or V158/F158 NK cells (C, D) is comparable to theADCC activity of the afucosylated antibody (3649-aFuc) mediated byF158/F158 NK cells (E).

FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D. Identification scheme of (A)circulating, (B) splenic, (C) bone marrow, and (D) peritoneal B cellsubsets based on cell surface antigen expression phenotype.Fluorescently stained isolated cell populations were analyzed on a flowcytometer. B cell subsets were identified and measured through thesequential use of gates. The flow of the process is indicated by bold,grey arrows. For example, follicular B cells of the spleen wereidentified as follows: (i) live cells are gated based on low 7AADstaining, (ii) lymphocytes from the live cell fraction are identifiedbased on their characteristic FSC and SSC phenotype, (iii) B cells amonglive lymphocytes are identified using anti-mCD19 and anti-B220 staining,(iv) B1a cells are separated from B cells based on differences in B220expression, (v) mature and transitional B cell population aredistinguished from each other based on differential expression of CD93,(vi) the follicular B cell subpopulation of mature B cells is separatedfrom the marginal zone B cell fraction based on differences in CD23expression.

FIG. 18. Binding profile of affinity matured 3649 anti-CD19 antibody Fabfragments to recombinant human CD19 expressing 300B4 cells in a cellbased ELISA assay. Results obtained with a representative sample of Fabscomprising single amino acid substitutions in the VH CDR3 is shown. The3649 anti-CD19 Fab (3649 peri) was used as reference standard. Theaffinity of 4G6 and 4B7 Fabs to recombinant human CD19 expressing 300B4cells is significantly higher than that of the control 3649 Fab. Allother Fabs tested have affinities similar to that of the control 3649Fab.

FIG. 19. Binding profile of affinity matured 3649 anti-CD19 antibody Fabfragments to recombinant human CD19 expressing 300B4 cells in a cellbased ELISA assay. The Fabs characterized here were identified from alibrary comprising all possible combinations of the beneficial singleamino acid substitutions identified in previous CDR specific screens.The binding profile of six Fabs with the highest affinity forrecombinant human CD19 expressing 300B4 cells is shown. The 3649anti-CD19 Fab (3649 peri) was used as reference standard. The affinityfor recombinant human CD19 expressing 300B4 cells of all six affinitymatured Fabs is higher than that of the control 3649 Fab.

FIG. 20. Binding profile of affinity matured 3649 anti-CD19 antibodiesto recombinant human CD19 expressing 300B4 cells in a cell based ELISAassay. The 3649 anti-CD19 antibody was used as reference standard. Thebinding profile of 16C9 IgG is similar to that of the 3649 controlantibody. The binding affinity of 14H5, 15D1, 15D7, 16C4, and 7E12affinity matured antibodies is higher than that of the control 3649antibody.

FIG. 21. Binding profile of affinity matured 3649 anti-CD19 antibodiesto CD19 expressing Raji cells in a cell based ELISA assay. The 3649anti-CD19 antibody was used as reference standard. The binding affinityof all six antibodies (14H5, 15D1, 15D7, 16C4, 16C9 and 7E12) is higherthan that of the control 3649 antibody.

FIG. 22. Binding profile of affinity matured 3649 anti-CD19 antibodiesto CD19 expressing Daudi cells in a cell based ELISA assay. The 3649anti-CD19 antibody was used as reference standard. The binding affinityof all six antibodies (14H5, 15D1, 15D7, 16C4, 16C9 and 7E12) is higherthan that of the control 3649 antibody.

FIG. 23. Binding profile of affinity matured 3649 anti-CD19 antibodiesto recombinant human CD19 expressing 300B4 cells in a cell based ELISAassay. 14H5-YG, 14H5-DG, and 14H5-LG are single amino acid substitutionvariants of the 14H5 affinity matured 3649 anti-CD19 antibody. 14H4 and16C4 affinity matured 3649 anti-CD19 antibodies were used as referencestandard. The binding affinity of 14H5-YG, 14H5-DG, and 14H5-LGantibodies is lower than that of the 14H5 and 16C4 control antibodies.

FIG. 24A and FIG. 24B. Kinetic off rate comparison of affinity matured3649 anti-CD19 antibodies. (A) Ramos cells were incubated with affinitymatured anti-CD19 antibodies, washed and further incubated at 37° C. for0, 30, or 60 minutes. Cells were stained with a fluorescent secondaryantibody at the end of the incubation period and analyzed on a flowcytometer. Mean fluorescent intensity of cells after 0, 30, and 60minute incubation is shown. The mean fluorescence intensity (MFI)observed at time 0 is set to 100% for each antibody studied. The 3649anti-CD19 antibody and an anti-CD20 antibody were used as referencestandards. The elimination of all six affinity matured 3649 anti-CD19antibodies (14H5, 15D1, 15D7, 16C4, 16C9 and 7E12) from the cell surfaceis slower than that of the reference standards. (B) Ramos cells werestained with an Alexa 647 conjugated HB12B, 3649, or 16C4 anti-CD19antibody, washed, and further incubated at 37° C. for 0, 30 or 60minutes. Cells were analyzed on flow cytometer at the end of theincubation period. A directly conjugated anti-CD20 antibody was includedin the experiment as a reference control. Mean fluorescence intensity(MFI) detected after various incubation periods is expressed as theratio of MFI seen at time 0. Loss of MFI after staining with the 16C4affinity matured anti-CD19 antibody is much slower than the loss ofsignal seen with the 3649 and HB12B anti-CD19 antibodies.

FIG. 25. Binding profile of affinity matured 3649 anti-CD19 antibodiesto Daudi cells. Daudi cells were stained with the 14H5, 15D1, 15D7,16C4, 16C9 or 7E12 affinity matured anti-CD19 antibodies and afluorescently labeled secondary antibody. The 3649 anti-CD19 antibodywas used as reference standard. Stained cells were analyzed on a flowcytometer. Median fluorescence intensity (Median FI) observed at variousantibody concentrations is presented in a chart. The median FI for theaffinity matured 3649 anti-CD-19 antibodies was higher than that of thereference standard.

FIG. 26A/B. In vitro ADCC activity of affinity matured 3649 anti-CD19antibodies. (A) In vitro ADCC activity of the 14H5, 14H5-DG and 16C4affinity matured anti-CD19 antibodies was assayed using Daudi targetcells. The 3649 anti-CD-19 antibody was used as reference standard. TheADCC activity of all three affinity matured antibodies is higher thanthat of the reference standard at low antibody concentration (0.01 and0.1 μg/ml antibody). The ADCC activity of all three affinity maturedantibodies parallels the activity of the reference standard at highantibody concentrations (1 and 10 μg/ml antibody). (B) The ADCC activityof the afucosylated 16C4 antibody (16C4-aFuc) was determined in an invitro assay using Daudi target cells. The ADCC activity of 16C4-aFuc issignificantly higher than that of the reference control 3649-aFuc,anti-CD20 and fucosylated 16C4 reference antibodies.

FIG. 27. Coomassic stained IEF-PAGE of affinity matured anti-CD19antibodies. The isoelectric point of the 16C4, 16C9, 7E12, 14H5, 15D7,15D1, 14H5-DG, and 3649 antibodies is 7.83, 8.04, 7.69, 7.76, 7.61,7.72, 7.48, and 7.75, respectively.

FIG. 28A and FIG. 28B. In vivo B cell depletion by the afucosylated 3649anti-CD19 antibody. C57B16 hCD19 tg+/− animals were treated with asingle i.v. dose of 10, 50, or 250 μg of fucosylated 3649 anti-CD19antibody (3649) or afucosylated 3649 anti-CD19 antibody (3649-aFuc).Negative control animals were treated with (i) the ADCC compromised Fcvariant of 3649 anti-CD19 antibody (3649 TM) or (ii) an antibody ofirrelevant specificity (R347). Circulating lymphocytes (A) or spleniclymphocytes (B) were isolated 7 days after antibody treatment. Isolatedcells were immunostained as described in Table 5 to identify various Bcell populations. Percentage of B220+ CD19+ B cells is displayed. Theafucosylated 3649 anti-CD19 antibody achieves a significantly higherdepletion of B cells than the same amount of fucosylated anti-CD19antibody. No depletion B cell depletion is detected in the 3649TMcontrol antibody treated animals.

FIG. 29. Enhanced NK cell activation in afucosylated 3649 anti-CD19antibody treated mice. C57B16 hCD19 tg+/− animals were treated with asingle i.v. dose of 10 μg of fucosylated 3649 anti-CD19 antibody (3649)or afucosylated 3649 anti-CD19 antibody (3649-aFuc). Negative controlanimals were treated with the same amount of an isotype matched antibodyof irrelevant specificity (R347). Circulating lymphocytes were isolated7 days after antibody treatment. Isolated cells were stained withfluorescently labeled anti-NK1.1, anti-DX5, and anti-CD107a antibodies.CD107a vs. NK1.1 plot of NK1.1+, DX5+ gated live lymphocytes isdisplayed. A higher percentage of NK cells isolated from afucosylated3649 anti-CD19 antibody treated animals display CD107a on their cellsurface than NK cells isolated from fucosylated 3649 anti-CD19 antibodytreated animals.

FIG. 30. Afucosylated anti-CD19 antibody #2 (3649-aFuc) significantlyreduces tumor growth in an in vivo model system. CB17 SCID mice wereinjected s.c. on the hind flank with 5×106 Raji cells on dayl. Animalswere treated with five biweekly doses of 10 mg/kg or 2.5 mg/kg antibodystarting on day 4. Antibodies used are: (i) fucosylated anti-CD19 #2 at10 mg/kg (3649), (ii) afucosylated anti-CD19 #2 at 10 mg/kg or 2.5 mg/kg(3649-aFuc), (iii) anti-CD20 at 10 mg/kg, and (iv) isotype controlantibody of irrelevant specificity at 10 mg/kg (R347). A group ofcontrol animals were only given PBS. Tumor size was measured twice aweek using standard procedures.

FIG. 31A and FIG. 31B. In vivo B cell depletion using the 16C4 and 14H5affinity mature anti-CD19 antibodies. C57B16 hCD19 tg+/− animals weretreated with a single i.v. dose of 10, 50, or 250 jug of fucosylated16C4 affinity matured anti-CD19 antibody (16C4) or 14H5DG affinitymatured anti-CD19 antibody (14H5DG). Reference control animals weretreated with (i) 3649 anti-CD19 antibody (3649), (ii) ADCC enhanced Fcvariant of 3649 anti-CD19 antibody (3649 3M), and (iii) afucosylated3649 anti-CD19 antibody (3649-aFuc). Negative control animals weretreated with (i) the ADCC compromised Fc variant of 3649 anti-CD19antibody (3649 TM) or (ii) an antibody of irrelevant specificity (R347).Circulating lymphocytes (A) or splenic lymphocytes (B) were isolated 7days after antibody treatment. Isolated cells were immunostained asdescribed in Table 5 to identify various B cell populations. Percentageof B220+ CD19+ B cells is displayed. The 16C4 affinity matured anti-CD19antibody achieved a slightly higher depletion of B cells than the 3649anti-CD19 parent antibody. The 3649-aFuc and 3649 3M antibodies achievedbetter depletion than the 16C4 affinity matured antibody. The 14H5DGaffinity matured anti-CD19 antibody is less efficient at depleting Bcells than the 3649 anti-CD19 parent antibody. Values inside panel (A)are that of the percent depletion achieved by a given antibody.

FIG. 32. Binding activity of the 64D4 affinity matured Fab torecombinant human CD19 expressing 300B4 cells in a cell based bindingassay (Lu et al., J. Immunol. Methods 314:74-79 (2006)). 64D4 is avariant of the 16C4 anti-CD19 antibody comprising a single amino acidsubstitution in the VH CDR2. The 16C4 and 3649 anti-CD19 Fabs (16C4supand 3649sup, respectively) were used as reference standards. Theaffinity of the 64D4 Fab to recombinant human CD19 expressing 300B4cells is significantly higher than that of the control 16C4 and 3649Fabs.

FIG. 33A, FIG. 33B and FIG. 33C. Characterization of the affinitymatured variant anti-CD19 Fabs isolated from the combinatorial phagedisplay library. Binding profile of affinity matured variants of the16C4 Fab to cell surface displayed human CD19 antigen was measure in acell based binding assay using (A) 300B4 and (B) Raji cells (Lu et al.,J. Immunol. Methods 314:74-79 (2006)). The 16C4 and 3649 anti-CD19 Fabs(16C4sup and 3649sup, respectively) were used as reference standards.The binding affinity of 6C11, 2B11, 3B4, 5C11, 3C3, 9G7, 1H4, and 5C4affinity matured Fabs to 300B4 and Raji cells is higher than that of thecontrol 3649 and 16C4 Fabs. (C) Amino acid sequence of the affinitymatured Fab clones was determined using standard laboratory methods. CDRsequence of unique Fab clones is presented. Amino acid residuesdifferent from that of the parental 16C4 sequence are printed usingsingle letter amino acid codes; residues identical to the parentalsequence are marked with a “−”. FIG. 33C discloses the “CDR1H” sequencesas SEQ ID NOS 22, 208, 208, 208, 208, 208, 208, 208, 22, 22, 208, 209,208, 22, 22, 208, 208, 208 and 208, respectively in order of appearance,the “CDR2H” sequences as SEQ ID NOS 116, 116, 116, 116, 116, 210, 116,116, 116, 116, 116, 116, 116, 116, 116, 210, 116, 116 and 116,respectively, in order of appearance, the “CDR3H” sequences all as SEQID NO: 121, the “CDR1L” sequences as SEQ ID NOS 28, 28, 211, 28, 28,212, 28, 28, 213, 28, 28, 28, 212, 214, 28, 28, 215, 239 and 217,respectively, in order of appearance, the “CDR2L” sequences as SEQ IDNOS 125, 125, 218, 125, 125, 125, 125, 125, 218, 125, 125, 125, 219 125,125, 220, 221, 218 and 218, respectively, in order of appearance, andthe “CDR3L” sequences as SEQ ID NOS 32, 32, 222-224, 32, 225, 226, 32,227, 228, 32, 224, 32, 226, 229, 222, 32 and 225, respectively, in orderof appearance.

FIG. 34. Binding profile of the 2B11, 3C3, 5C4, 6C11, 6F7, and 9G7affinity matured IgG anti-CD19 antibodies to recombinant human CD19expressing 300B4 cells. Binding activity was measured using a cell basedassay (Lu et al., J. Immunol. Methods 314:74-79 (2006)). The 16C4 and3649 anti-CD19 antibodies were used as reference standards. The bindingaffinity of the affinity matured anti-CD19 antibodies tested to 300B4cells is higher than that of the reference 16C4 and 3649 antibodies.

FIG. 35A and FIG. 35B. Binding profile of affinity matured 16C4anti-CD19 antibodies to (A) Raji cells and (B) Daudi cells. Cells werestained with the 3C3, 6C11, or 9G7 affinity matured anti-CD19 antibodiesand a fluorescently labeled secondary antibody. The 16C4 anti-CD19antibody was used as reference standard. Stained cells were analyzed ona flow cytometer. Median fluorescence intensity (Median FI) observed atvarious antibody concentrations is presented. The median FI of cellsstained with the affinity matured 16C4 variant anti-CD19 antibodies washigher than that of the reference standard stained cells at 0.0625-0.125μg/ml primary antibody concentration. Median FI obtained using affinitymatured antibodies was substantially the same as that of for thereference antibody in the 0.25-10 μg/ml range.

FIG. 36. In vitro ADCC activity of affinity matured 16C4 variantanti-CD19 antibodies. In vitro ADCC activity of the 3C3, 6C11, or 9G7affinity matured anti-CD19 antibodies was assayed using Raji targetcells. The 16C4 anti-CD19 antibody was used as reference standard. TheADCC activity of all three affinity matured antibodies is substantiallythe same as that of the reference standard at all concentrations tested(0.01-10 μg/ml).

FIG. 37. In vitro ADCC activity of affinity matured 16C4 variantanti-CD19 antibodies. In vitro ADCC activity of the 3C3, 6C11, or 9G7affinity matured anti-CD19 antibodies was assayed using Daudi targetcells. The 16C4 anti-CD-19 antibody was used as reference standard. TheADCC activity of all three affinity matured antibodies is substantiallythe same as that of the reference standard at all concentrations tested(0.01-10 μg/ml).

FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, FIG. 38E, FIG. 38F and FIG. 38G.Long term recovery of B cells and serum immunoglobulin levels followingB cell depletion with a single dose of i.v. administered afucosylated16C4 anti-CD19 antibody. (A) Experimental protocol. Groups of four orfive huCD19 tg+/− mice were administered a single i.v. dose of 250, 50,or 10 μg afucosylated 16C4 anti-CD19 antibody (16C4 aFuc). Controlgroups were treated with either PBS or 250 μs control antibody ofirrelevant specificity (R347). Animals were bled once every two weeks;first bleed was done seven days prior to administration of the depletingantibody. Findings from the first 11 weeks are summarized in the panels.(B) The body weight of the animals in all groups remained normal. BloodB cell levels are expressed as (C) the fraction of lymphocytes or as (D)B cell number per microliter of blood. All three 16C4 aFuc antibodydoses achieved complete B cell depletion. B cell recovery was completeby week 5 and week 9 in animals receiving 10 and 50 16C4 aFuc antibody,respectively. B cell recovery was still incomplete 11 weeks after theadministration of 250 μg 16C4 aFuc antibody. Serum (E) IgM, (F) IgG1,and (G) IgG2b was unchanged following the administration of 50 or 250 μg16C4 aFuc. Serum immunoglobulin levels were increased following theadministration of 10 μg 16C4 aFuc antibody or the control antibody R347or PBS. The data indicates that 16C4 aFuc suppressed on-goingimmunoglobulin production, but had minimum impact on the pre-existingimmunoglobulin in serum.

FIG. 39A-B and FIG. 39C-D. Anti-CD19 antibody induced intracellularsignaling. (A-B) 3649, 3649-TM, 3649-3M, 3649-aFuc, or 16C4 antibodytreatment significantly increases the tyrosine phosphorylation level ofCD19 in Raji cells. (C-D) Anti-CD19 antibody treatment does not inhibitanti-IgM treatment induced ERK1/2 phosphorylation.

FIG. 40. Anti-CD19 antibody treatment inhibits anti-IgM/CD40 mediated Bcell proliferation.

FIG. 41A and FIG. 41B. Anti-CD19 antibody treatment inhibits theanti-IgM/CpG induced proliferation of purified peripheral B cells. (A)Fluorescence intensity profile of CFSE stained purified peripheral bloodB cells following 4 days of incubation in the presence anti-IgM (1μg/ml) and CpG (2 μg/ml). The CFSE profiles of an unstimulated firstcontrol cell population and a CpG only stimulated second control cellpopulation are included as reference standards. (B) CFSE profiles of Bcells following 4 days of stimulation with anti-IgM/CpG in the presenceof 16C4 anti-CD19 or R347 control antibody. Anti-IgM/CpG induced B cellproliferation is significantly reduced in the presence of 16C4 antibody.

FIG. 42A-C. The 3649-3M Fc variant anti-CD19 antibody is a moreeffective inhibitor of anti-IgM/CpG induced B cell proliferation thanthe 3649-TM Fc variant antibody. CF SE profiles of B cells following 4days of stimulation by anti-IgM/CpG in the presence of (A) R347 control,(B) 3649-TM anti-CD19, or (C) 3649-3M anti-CD19 antibody.

FIG. 43A-D. 3649-3M antibody induced signaling through the CD19 andFcgammaRIIB receptors synergistically inhibits anti-IgM/CpG mediated Bcell proliferation.

FIG. 44. Surface bound anti-CD19 antibody is efficiently internalized byRaji cells. 35% of surface bound 16C4 and 55% of surface bound HB12B and3649 anti-CD19 antibody is internalized following 60 minutes incubationat 37° C.

FIG. 45A-B. Surface expression of CD19 is significantly reducedfollowing 24 hours of anti-CD19 antibody treatment. Cell surfaceexpression of CD19 is reduced by 55-90% in (A) Raji cells and (B)purified peripheral B cells following 24 hours incubation in thepresence of 3649, 3649-TM, 3649-3M, 3649-aFuc, or 16C4 anti-CD19antibody.

5. DETAILED DESCRIPTION

The present invention relates to human, humanized, or chimeric anti-CD19antibodies that bind to the human CD19 antigen, as well as tocompositions comprising those antibodies. In certain embodiments ahuman, humanized, or chimeric anti-CD19 antibody may mediateantigen-dependent-cell-mediated-cytotoxicity (ADCC). In otherembodiments, the present invention is directed toward compositionscomprising a human, humanized, or chimeric anti-CD19 antibody of theIgG1 and/or IgG3 human isotype, as well as to a human, humanized, orchimeric anti-CD19 antibody of the IgG2 and/or IgG4 human isotype, thatmay mediate human ADCC, CDC, and/or apoptosis. In further embodiments ahuman, humanized, or chimeric anti-CD19 antibody may inhibitanti-IgM/CpG stimulated B cell proliferation.

The present invention provides chimeric and humanized versions of theanti-CD19 mouse monoclonal antibodies HB12A and HB12B. In oneembodiment, a humanized anti-CD19 antibody of the invention may bind tohuman CD19 with an affinity comparable to the binding affinity of HB12Aor HB12B or comparable to the binding affinity of a chimeric HB12Bantibody.

In one embodiment, a humanized anti-CD19 monoclonal antibody of theinvention may comprise a VH and a VK, wherein the VH comprises the fourframework regions, FW1, FW2, and FW3 of the human germline VH segment ofV3-72 (described as DP29 in Tomlinson, I. M. et al., (1992) J Mol.Biol., 227, 776-798), and FW4 of the human germline JH4 segment(Mattila, P. S. et al., (1995) Eur. J. Immunol., 25, 2578-2582); and thethree VH CDR sequences of the HB12B antibody, CDR1 (SEQ ID NO:22), CDR2(SEQ ID NO:24), and CDR3 (SEQ ID NO:26); and the VK comprises the fourframework regions, FW1, FW2, FW3 of the human germline V kappa segmentA10 (Straubinger, B. I et al., (1988) Biol. Chem. Hoppe-Seyler, 369,601-607), and FW4 of the human germline immunoglobulin kappa J4 segment(Hieter, P. A. et al., (1982) J. Biol. Chem., 257, 1516-1522); and thethree VK CDR sequences of the HB12B antibody, CDR1 (SEQ ID NO:28), CDR2(SEQ ID NO:30), and CDR3 (SEQ ID NO:32). In one embodiment, an anti-CD19antibody of the invention may comprise a VH and a VK, wherein the VHcomprises the four framework regions, FW1, FW2, and FW3 of the humangermline VH segment of V3-72 (described as DP29 in Tomlinson, I. M. etal., (1992) J Mol. Biol., 227, 776-798), and FW4 of the human germlineJH4 segment (Mattila, P. S. et al., (1995) Eur. J. Immunol., 25,2578-2582); and at least one CDR having the amino acid sequence of a CDRlisted on Table 1 supra; and the VK comprises the four frameworkregions, FW1, FW2, FW3 of the human germline V kappa segment A10(Straubinger, B. I et al., (1988) Biol. Chem. Hoppe-Seyler, 369,601-607), and FW4 of the human germline immunoglobulin kappa J4 segment(Hieter, P. A. et al., (1982) J. Biol. Chem., 257, 1516-1522); and atleast one CDR having the amino acid sequence of a CDR listed on Table 1supra. In one embodiment, this antibody may comprise one or more VKframework mutations selected from the group consisting of Y40F, K53H andY91F. In one embodiment, the VK framework region may contain each of thepoint mutations Y40F, K53H and Y91F. In another embodiment, the VKframework region may contain only the Y4OF and K53H point mutations. Inanother embodiment the VK framework may comprise only the Y4OF pointmutation.

5.1.1. CDR Regions of Anti-CD19 Antibodies

In certain embodiments, an anti-CD19 antibody of the invention maycomprise a heavy chain variable region, VH, comprising at least one CDRhaving the amino acid sequence selected from the group consisting of SEQID NO:22, SEQ ID NO:24, and SEQ ID NO:26; and may further comprise atleast one FW region having the amino acid sequence selected from thegroup consisting of SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, and SEQ IDNO:42. In another embodiment, an anti-CD19 antibody of the invention maycomprise a heavy chain variable region, VH, comprising at least one CDRhaving the amino acid sequence selected from the group consisting of SEQID NO:22, SEQ ID NO:24, and SEQ ID NO:121 and may further comprise atleast one FW region having the amino acid sequence selected from thegroup consisting of SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, and SEQ IDNO:42. In a further embodiment, an anti-CD19 antibody of the inventionmay comprise a heavy chain variable region, VH, comprising at least oneCDR having the amino acid sequence selected from the group consisting ofSEQ ID NO:22, SEQ ID NO:116, and SEQ ID NO:121 and may further compriseat least one FW region having the amino acid sequence selected from thegroup consisting of SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, and SEQ IDNO:42. In a further embodiment, an anti-CD19 antibody of the inventionmay comprise a heavy chain variable region, VH, comprising at least oneCDR having the amino acid sequence selected from the group consisting ofSEQ ID NO:208, SEQ ID NO:116, and SEQ ID NO:121 and may further compriseat least one FW region having the amino acid sequence selected from thegroup consisting of SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, and SEQ IDNO:42. In a further embodiment, an anti-CD19 antibody of the inventionmay comprise a heavy chain variable region, VH, comprising at least oneCDR having the amino acid sequence selected from the group consisting ofSEQ ID NO:208, SEQ TD NO:210, and SEQ ID NO:121 and may further compriseat least one FW region having the amino acid sequence selected from thegroup consisting of SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, and SEQ IDNO:42. In another embodiment, an anti-CD19 antibody of the invention maycomprise a heavy chain variable region, VH, comprising at least one CDRhaving the amino acid sequence of a VH CDR1, VH CDR2, or VH CDR3 listedin Table 1 supra; and may further comprise at least one FW region havingthe amino acid sequence selected from the group consisting of SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, and SEQ ID NO:42.

In further embodiments, an anti-CD19 antibody of the invention maycomprise a heavy chain variable region, VH, comprising at least one CDRsequence selected from the group consisting of SEQ ID NO:22, SEQ IDNO:24, and SEQ ID NO:26.

In additional embodiments, an anti-CD19 antibody may comprise a heavychain variable region, VH, comprising at least one CDR sequence selectedfrom the group consisting of SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10.

In one embodiment, an anti-CD19 antibody of the invention may comprise aheavy chain variable region, VH, comprising at least one CDR having theamino acid sequence selected from the group consisting of SEQ ID NO:22,SEQ ID NO:24 and SEQ ID NO:121. In another embodiment, an anti-CD19antibody of the invention may comprise a heavy chain variable region,VH, comprising at least one CDR having the amino acid sequence selectedfrom the group consisting of SEQ ID NO:22, SEQ ID NO:116 and SEQ IDNO:121. In another embodiment, an anti-CD19 antibody of the inventionmay comprise a heavy chain variable region, VH, comprising at least oneCDR having the amino acid sequence selected from the group consisting ofSEQ ID NO:208, SEQ ID NO:116 and SEQ ID NO:121. In another embodiment,an anti-CD19 antibody of the invention may comprise a heavy chainvariable region, VH, comprising at least one CDR having the amino acidsequence selected from the group consisting of SEQ ID NO:208, SEQ IDNO:210 and SEQ ID NO:121.

In another embodiment, an anti-CD19 antibody of the invention maycomprise a heavy chain variable region, VH, comprising at least one CDRhaving the amino acid sequence of a VH CDR1, VH CDR2, or VH CDR3 listedin Table 1 supra.

In another embodiment, an anti-CD19 antibody of the invention maycomprise a heavy chain variable region, VH, comprising the amino acidsequences of a VH CDR1, VH CDR2, and VH CDR3 of any one of theantibodies listed in Table 1 supra. The anti-CD19 antibody of theinvention may further comprise a light chain variable region, VL.

In another embodiment, an anti-CD19 antibody of the invention maycomprise a light chain variable region, VL, comprising the amino acidsequences of a VL CDR1, VL CDR2, and VL CDR3 of any one of theantibodies listed in Table 1 supra. The anti-CD19 antibody of theinvention may further comprise a heavy chain variable region, VH.

In another embodiment, an anti-CD19 antibody of the invention maycomprise the amino acid sequences of a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 of any one of the antibodies listed in Table1 supra.

In certain embodiments, an anti-CD19 antibody may comprise the VH domainsequence of the humanized VH designated HB12B-(3-72/JH4) (SEQ ID NO:34).

In one embodiment, an anti-CD19 antibody described herein may comprise aheavy chain variable region, VH, having the amino acid sequence selectedfrom the group consisting of SEQ ID NOs:103, 106, 191, and 192. Inanother embodiment, an anti-CD19 antibody described herein may comprisea heavy chain variable region, VH, having the amino acid sequence of aVH Domain listed in Table 1. supra.

In certain embodiments, an anti-CD19 antibody of the invention maycomprise a light chain variable region, VK, comprising at least one CDRhaving an amino acid sequence selected from the group consisting of SEQID NO: 28, SEQ ID NO: 30, and SEQ ID NO:32 and may further comprise atleast one FW region having an amino acid sequence selected from thegroup consisting of SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:72, SEQ IDNO:82, SEQ ID NO:64, SEQ ID NO:58, SEQ ID NO:66, and SEQ ID NO:60.

In one embodiment, an anti-CD19 antibody of the invention may comprise alight chain variable region, VK, comprising at least one CDR having anamino acid sequence selected from the group consisting of SEQ ID NO: 28,SEQ ID NO: 125, and SEQ ID NO:32 and may further comprise at least oneFW region having an amino acid sequence selected from the groupconsisting of SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:72, SEQ ID NO:82,SEQ ID NO:64, SEQ ID NO:58, SEQ ID NO:66, and SEQ ID NO:60. In a furtherembodiment, an anti-CD19 antibody of the invention may comprise a lightchain variable region, VK, comprising at least one CDR having an aminoacid sequence selected from the group consisting of SEQ ID NO:211, SEQID NO:218, and SEQ ID NO:222 and may further comprise at least one FWregion having an amino acid sequence selected from the group consistingof SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:64,SEQ ID NO:58, SEQ ID NO:66, and SEQ ID NO:60. In a further embodiment,an anti-CD19 antibody of the invention may comprise a light chainvariable region, VK, comprising at least one CDR having an amino acidsequence selected from the group consisting of SEQ ID NO:28, SEQ IDNO:220, and SEQ ID NO:229 and may further comprise at least one FWregion having an amino acid sequence selected from the group consistingof SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:64,SEQ ID NO:58, SEQ ID NO:66, and SEQ ID NO:60. In a further embodiment,an anti-CD19 antibody of the invention may comprise a light chainvariable region, VK, comprising at least one CDR having an amino acidsequence selected from the group consisting of SEQ ID NO:215, SEQ IDNO:221, and SEQ ID NO:222 and may further comprise at least one FWregion having an amino acid sequence selected from the group consistingof SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:64,SEQ ID NO:58, SEQ ID NO:66, and SEQ ID NO:60. In another embodiment, ananti-CD19 antibody of the invention may comprise a light chain variableregion, VK, comprising at least one CDR having an amino acid sequence ofa VK CDR1, VK CDR2, or VK CDR3 listed in Table 1 supra; and may furthercomprise at least one FW region having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:72,SEQ ID NO:82, SEQ ID NO:64, SEQ ID NO:58, SEQ ID NO:66, and SEQ IDNO:60.

In further embodiments, an anti-CD19 antibody of the invention maycomprise a light chain variable region, VK, comprising at least one CDRsequence selected from the group consisting of SEQ ID NO:28, 30, and 32.

In further embodiments, an anti-CD19 antibody of the invention maycomprise a light chain variable region, VK, comprising at least one CDRsequence selected from the group consisting of SEQ ID NO:12, 14, and 16.

In one embodiment, an anti-CD19 antibody of the invention may comprise alight chain variable region, VK, comprising at least one CDR having anamino acid sequence selected from the group consisting of SEQ ID NO: 28,SEQ ID NO: 125, and SEQ ID NO:32. In one embodiment, an anti-CD19antibody of the invention may comprise a light chain variable region,VK, comprising at least one CDR having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:211, SEQ ID NO: 218, and SEQ IDNO:222. In one embodiment, an anti-CD19 antibody of the invention maycomprise a light chain variable region, VK, comprising at least one CDRhaving an amino acid sequence selected from the group consisting of SEQID NO: 28, SEQ ID NO:220, and SEQ ID NO:229. In one embodiment, ananti-CD19 antibody of the invention may comprise a light chain variableregion, VK, comprising at least one CDR having an amino acid sequenceselected from the group consisting of SEQ ID NO: 215, SEQ ID NO: 221,and SEQ ID NO:222. In another embodiment, an anti-CD19 antibody of theinvention may comprise a light chain variable region, VK, comprising atleast one CDR having an amino acid sequence of a VK CDR1, VK CDR2, or VKCDR3 listed in Table 1 supra.

In certain embodiments, an anti-CD19 antibody may comprise the humanizedVK domain sequence selected from a group consisting of HB12B-(A10-Jk4)(SEQ ID NO:52), HB12B-364987 (SEQ ID NO:62), HB12B-3649 (SEQ ID NO:68),HB12B-36 (SEQ ID NO:70), 7E12 VK (SEQ ID NO:110), 14H5 VK (SEQ IDNO:111), 16C9 VK (113), 15D1 VK (SEQ ID NO:112), 3C3 VK (SEQ ID NO:193),6C11 VK (SEQ ID NO:204), and 9G7 VK (SEQ ID NO:205).

The present invention encompasses antibodies that bind to human CD19,comprising derivatives of the VH domains, VH CDR1s, VH CDR2s, VH CDR3s,VK domains, VK CDR1s, VK CDR2s, or VK CDR3s described herein that maybind to human CD19 (see for example the variants listed in Table 1.supra). Standard techniques known to those of skill in the art can beused to introduce mutations (e.g., additions, deletions, and/orsubstitutions) in the nucleotide sequence encoding an antibody,including, for example, site-directed mutagenesis and PCR-mediatedmutagenesis that are routinely used to generate amino acidsubstitutions. In one embodiment, the VH and/or VK CDRs derivatives mayinclude less than 25 amino acid substitutions, less than 20 amino acidsubstitutions, less than 15 amino acid substitutions, less than 10 aminoacid substitutions, less than 5 amino acid substitutions, less than 4amino acid substitutions, less than 3 amino acid substitutions, lessthan 2 amino acid substitutions, or 1 amino acid substitution relativeto the original VH and/or VK CDRs of the HB12A or HB12B anti-CD19antibody. In another embodiment, the VH and/or VK CDRs derivatives mayhave conservative amino acid substitutions (e.g. supra) made at one ormore predicted non-essential amino acid residues (i.e., amino acidresidues which are not critical for the antibody to specifically bind tohuman CD19). Mutations can also be introduced randomly along all or partof the VH and/or VK CDR coding sequences, such as by saturationmutagenesis, and the resultant mutants can be screened for biologicalactivity to identify mutants that retain activity. Followingmutagenesis, the encoded antibody can be expressed and the activity ofthe antibody can be determined. In one embodiment, antibodies of theinvention disclosed herein may exclude the VH CDR1 and VH CDR2 of thehA19 antibody described in US20050070693A1.

In one embodiment, a human or humanized anti-CD19 antibody describedherein may comprise a variant of any one of the VH CDRs listed in Table1 supra wherein said variant VH CDR comprises an amino acidsubstitution. In a specific embodiment, an anti-CD19 antibody of theinvention comprises a variant of a VH CDR listed in Table 1 wherein saidvariant VH CDR comprises one or more of the following natural orsubstituted amino acid residues: a threonine (T) at position 32 of VHCDR1, a tyrosine (Y) at position 60 of VH CDR2, an aspartic acid (D) atposition 60 of VH CDR2, a leucine (L) at position 60 of VH CDR2, analanine (A) at position 61 of VH CDR2, a valine (V) at position 61 of VHCDR2, a tyrosine (Y) at position 100B of VH CDR3, an arginine (R) atposition 100B of VH CDR3, and an asparagine (N) at position 100B of VHCDR3, numbered according to Kabat.

In one embodiment, a human or humanized anti-CD19 antibody describedherein may comprise a variant of a VH CDR listed in Table 1. whereinsaid variant VH CDR comprises one or more of the following natural orsubstituted amino acid residues: a glutamic acid (E) at position 33 ofVH CDR1, a leucine (L) at position 33 of VHCDR1, phenylalanine (F) atposition 35 of VH CDR1, a tyrosine (Y) at position 35 of VH CDR1, anaspartic acid (D) at position 35 of VH CDR1, a leucine (L) at position35 of VH CDR1, a serine (S) at position 57 of VH CDR2, a proline (P) atposition 57 of VH CDR2, an asparagine (N) at position 57 of VH CDR2, ahistidine (H) at position 100B of VH CDR3, a phenylalanine (F) atposition 100B of VH CDR3, and a proline (P) at position 99 of VH CDR3,numbered according to Kabat.

In one embodiment, a human or humanized anti-CD19 antibody describedherein may comprise a variant of a VH CDR listed in Table 1. whereinsaid variant VH CDR comprises one or more of the following natural orsubstituted amino acid residues: a valine (V) at position 32 of VH CDR1,and a leucine (L) at position 52A of VHCDR2, numbered according toKabat.

In another embodiment, a human or humanized anti-CD19 antibody of theinvention may comprise a variant of a VK CDR listed in Table 1 whereinsaid VK CDR comprises one or more of the following natural orsubstituted amino acid residues: a histidine (H) at position 27D of VKCDR1, an isoleucine (I) at position 33 of VK CDR1, a glutamic acid (E)at position 50 of VK CDR2, a threonine (T) at position 91 in VK CDR3,and an isoleucine (I) at position 96 of VK CDR3, numbered according toKabat.

In another embodiment, a human or humanized anti-CD19 antibody of theinvention may comprise a variant of a VK CDR listed in Table 1 whereinsaid VK CDR comprises one or more of the following natural orsubstituted amino acid residues: a isoleucine (1) at position 27C of VKCDR1, a leucine (L) at position 30 of VK CDR1, an arginine (R) atposition 33 of VK CDR1, a threonine (T) at position 33 of VK CDR1, atyrosine (Y) at position 50 of VK CDR2, a threonine (T) at position 54of VK CDR2, a proline (P) at position 54 of VK CDR2, a tyrosine (Y) atposition 55 of VK CDR2, and an asparagine (N) at position 96 of VK CDR3,numbered according to Kabat.

In another embodiment, a human or humanized anti-CD19 antibody of theinvention may comprise a variant of a VK CDR listed in Table 1 whereinsaid VK CDR comprises one or more of the following natural orsubstituted amino acid residues: an arginine (R) at position 54 of VKCDR2, a thrconinc (T) at position 54 of VK CDR2, an alaninc (A) atposition 54 of VK CDR2, and an alanine (A) at position 89 of VK CDR3,numbered according to Kabat.

The present invention further encompasses antibodies that bind to humanCD19, said antibodies or antibody fragments comprising one or more CDRswherein said CDRs comprise an amino acid sequence that is at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to the amino acid sequence of one or more CDRs ofthe HB12A or HB12B anti-CD19 antibody. The percent identity of two aminoacid sequences can be determined by any method known to one skilled inthe art, including, but not limited to, BLAST protein searches.

5.1.2. Framework Regions of Anti-CD19 Antibodies

In one embodiment, the VH of a humanized anti-CD19 monoclonal antibodyof the invention may comprise a framework region that has an amino acidsequence identity with the corresponding framework regions (i.e., FW1 ofantibody X as compared to FW1 of antibody Y) of HB12B-(3-72/JH4) VH (SEQID NO:34) within the range of from about 64% to about 100%. In certainaspects of this embodiment, the human or humanized VH framework regionsof antibodies described herein may have an amino acid sequence identitywith the HB12B-(3-72/JH4) VH (SEQ ID NO:34) that is at least 64%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%.

In particular embodiments, the human or humanized VH framework regionsof anti-CD19 antibodies described herein may have an amino acid sequenceidentity with the corresponding framework regions of HB12B-(3-72/JH4) VH(SEQ ID NO:34) of at least 56 out of 87 amino acids (56/87) Inparticular embodiments, the VH framework amino acid sequence identitymay be at least 56/87, 57/87, 58/87, 59/87, 60/87, 61/87, 62/87, 63/87,64/87, 65/87, 66/87, 67/87, 68/87, 69/87, 70/87, 71, 87, 72/87, 73/8774/87, 75/87, 76/87, 77.87, 78/87, 79/87, 80/87, 81/87, 82/87, 83/87,84/87, 85/87, 86/87, or 87/87 amino acids. VH sequences of anti-CD19antibodies described herein may have high sequence identity to theVernier amino acid residues of HB12B-(3-72/JH4), for example a Verniersequence identity of at least 10 out of 16 (10/16), at least 11/16, atleast 12/16, at least 13/16, at least 14/16, or at least 15/16 Vernierresidues. In another embodiment, the mismatch of a Vernier amino acidresidue may be a conservative amino acid substitution. A mismatch thatis a conservative amino acid substitution is one in which the mismatchedamino acid has physical and chemical properties similar to the Vernieramino acid, e.g., the mismatched residue has similar characteristics ofpolarity (polar or nonpolar), acidity (acidic or basic), side chainstructure (e.g., branched or straight, or comprising a phenyl ring, ahydroxyl moiety, or a sulfur moiety) to the Vernier residue.

In other embodiments, the mismatch of a Vernier amino acid residue maybe a non-conservative amino acid substitution. A mismatch that is anon-conservative amino acid substitution is one in which the mismatchedamino acid does not have physical and chemical properties similar to theVernier amino acid, e.g., the mismatched residue has a differentpolarity, acidity, or side chain structure (e.g., branched or straight,or comprising a phenyl ring, a hydroxyl moiety, or a sulfur moiety) ascompared to the Vernier residue to be replaced.

In other embodiments, a human or humanized anti-CD19 antibody of theinvention may comprise VH framework regions wherein said VH frameworkregions may comprise one or more of the following residues: a leucine(L) at position 20 of framework region 1, a phenylalanine (F) atposition 27 of framework region 1, a threonine (T) at position 28 offramework region 1, an arginine (R) at position 38 in framework region2, a valine (V) at position 48 of framework region 2, a phenylalanine(F) at position 67 of framework region 3, an arginine (R) at position 71of framework region 3, a leucine (L) at position 80 of framework region3, and a tyrosine (Y) at position 91 of framework region 3, numberedaccording to Kabat.

Kabat numbering is based on the seminal work of Kabat et al. (1991)Sequences of Proteins of Immunological Interest, Publication No.91-3242, published as a three volume set by the National Institutes ofHealth, National Technical Information Service (hereinafter “Kabat”).Kabat provides multiple sequence alignments of immunoglobulin chainsfrom numerous species antibody isotypes. The aligned sequences arenumbered according to a single numbering system, the Kabat numberingsystem. The Kabat sequences have been updated since the 1991 publicationand are available as an electronic sequence database (latestdownloadable version 1997). Any immunoglobulin sequence can be numberedaccording to Kabat by performing an alignment with the Kabat referencesequence. Accordingly, the Kabat numbering system provides a uniformsystem for numbering immunoglobulin chains. Unless indicated otherwise,all immunoglobulin amino acid sequences described herein are numberedaccording to the Kabat numbering system. Similarly, all single aminoacid positions referred to herein are numbered according to the Kabatnumbering system.

In further particular embodiments, the human or humanized VH frameworkregions of anti-CD19 antibodies described herein may have frameworkregions selected for identity or conservative mismatches at one or moreof the following Vernier, Interface or Canonical residue positions: 20,22, 24, 26, 27, 28, 29, 30, 36, 37, 39, 45, 47, 48, 49, 67, 69, 71, 73,78, 80, 90, 91, 92, 93, 94, and 103. One or more of the mismatchedVernier, Interface and Canonical residues may be changed, e.g., bymutagenesis, to match the corresponding amino acid residue of the HB12Aor HB12B VH framework region.

In one embodiment of the invention, the human or humanized VK frameworkregions of anti-CD19 antibodies described herein may have an amino acidsequence identity with the framework regions of HB12B-(A10-Jk4) VK (SEQID NO:52) within the range of from about 65% to about 100%. In certainaspects of this embodiment, the human or humanized VK framework regionsof antibodies described herein may have an amino acid sequence identitywith the HB12B-(A10-Jk4) antibody VK that is at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

In particular embodiments, the human or humanized VK framework regionsof antibodies described herein may have an amino acid sequence identitywith the corresponding framework regions (i.e., FW1 of antibody X ascompared to FW1 of antibody Y) of HB12B-(A10-Jk4) VH (SEQ ID NO:52) ofat least 52 out of 80 amino acids (52/80) In particular embodiments, theVH framework amino acid sequence identity may be at least 52/80, 53/8054/80, 55/80, 56/80, 57/80, 58/80, 59/80, 60/80, 61/80, 62/80, 63/80,64/80, 65/80, 66/80, 67/80, 68/80, 69/80, 70/80, 71, 80, 72/80, 73/8074/80, 75/80, 76/80, 77/80, 78/80, 79/80, or 80/80, amino acids. VKsequences of anti-CD19 antibodies described herein may have highsequence identity to the Vernier amino acid residues of HB12B (see FIG.1), for example a Vernier sequence identity of at least 9 out of 14(9/14), at least 10/14, at least 11/14, at least 12/14, at least 13/14Vernier residues. In another embodiment, the mismatch of a Vernier aminoacid residue may be a conservative amino acid substitution. A mismatchthat is a conservative amino acid substitution is one in which themismatched amino acid has physical and chemical properties similar tothe Vernier amino acid, e.g., the mismatched residue has similarcharacteristics of polarity (polar or nonpolar), acidity (acidic orbasic), side chain structure (e.g., branched or straight, or comprisinga phenyl ring, a hydroxyl moiety, or a sulfur moiety) to the Vernierresidue.

In other embodiments, the mismatch of a Vernier amino acid residue maybe a non-conservative amino acid substitution. A mismatch that is anon-conservative amino acid substitution is one in which the mismatchedamino acid does not have physical and chemical properties similar to theVernier amino acid, e.g., the mismatched residue has a differentpolarity, acidity, or side chain structure (e.g., branched or straight,or comprising a phenyl ring, a hydroxyl moiety, or a sulfur moiety) ascompared to the Vernier residue to be replaced.

In other embodiments, the human or humanized VK framework regionsdescribed herein may comprise one or more of the following residues: aphenylalaninc (F) at position 36 of framework region 2, a histidine (H)at position 49 of framework region 2, and a phenylalanine (F) atposition 87 of framework region 3, numbered according to Kabat.

In further particular embodiments, the human or humanized VK frameworkregions of antibodies described herein may have framework regionsselected for identity or conservative mismatches at one or more of thefollowing Vernier, Interface or Canonical residue positions: 2, 3, 4,23, 35, 36, 38, 44, 56, 47, 48, 49, 64, 66, 68, 69, 71, 87, 88, and 98.One or more of the mismatched Vernier, Interface and Canonical residuesmay be changed, e.g., by mutagenesis, to match the corresponding aminoacid residue of the HB12A or HB12B framework region.

In particular embodiments, a heavy chain comprising a humanized VH ofthe invention may be expressed with a light chain comprising a humanizedVK of the invention to produce a humanized anti-CD19 antibody. In aspecific embodiment, a humanized anti-CD19 antibody of the invention maycomprise a VH sequence selected from the group consisting of thesequences designated HB12B-(3-72/JH4) (SEQ ID NO:34), 7E12 VH (SEQ IDNO:102), 14H5 VH (SEQ ID NO:103), 15D1 VH (SEQ ID NO:104), 15D7 VH (SEQID NO:105), 16C4 VH (SEQ ID NO:106), 14H5-YG (SEQ ID NO:107), 14H5-DG(SEQ ID NO:108), 14H5-LG (SEQ ID NO:109), 1A7 VH (SEQ ID NO:191), 3C3 VH(SEQ ID NO:191), 6C11 VH (SEQ ID NO:191), 9G7 (SEQ ID NO:191), 3B4 VH(SEQ ID NO:236), and 3F11 VH (SEQ ID NO:192); and may further comprise aVK sequence selected from the group consisting of the sequencesdesignated HB12B-(A 1 0/JK4) (SEQ ID NO:52); HB12B-364987 (or 364987)(SEQ ID NO:62); HB12B-3649 (or 3649) (SEQ ID NO:68); HB12B-36 (or 36)(SEQ ID NO:70), 7E12 VK (SEQ ID NO:110), 14H5 (SEQ ID NO:111), 15D1 (SEQID NO:112), 16C9 (SEQ ID NO:113), 3C3 VK (SEQ ID NO:193), 3E5 VK (SEQ IDNO:194), 3D4 VK (SEQ ID NO:195), 3F1 VK (SEQ ID NO:196), 5B5 VK (SEQ IDNO:197), 6F7 VK (SEQ ID NO:198), 1C11 VK (SEQ ID NO:199), 2B11 VK (SEQID NO:200), 2D10 VK (SEQ ID NO:201), 5C11 VK (SEQ ID NO:202), 5D4 VK(SEQ ID NO:203), 6C11 VK (SEQ ID NO:204), 9G7 VK (SEQ ID NO:205), 1H4 VK(SEQ ID NO:206), and 5C4 VK (SEQ ID NO:207). In a particular embodiment,a humanized anti-CD19 antibody comprises the VH sequenceHB12B-(3-72/JH4) (SEQ ID NO:34) and the VK sequence HB12B-364987 (SEQ IDNO:62). In a particular embodiment, a humanized anti-CD19 antibodycomprises the VH sequence HB12B-(3-72/JH4) (SEQ ID NO:34) and the VKsequence HB12B-3649 (SEQ ID NO:68). In yet another embodiment, ahumanized anti-CD19 antibody comprises the VH sequence HB12B-(3-72/JH4)(SEQ ID NO:34) and the VK sequence HB12B-36 (SEQ ID NO:70).

In a specific embodiment, an anti-CD19 antibody of the inventioncomprises the VH sequence 7E12 VH (SEQ ID NO:102) and the VK sequence7E12 VK (SEQ ID NO:110). In a specific embodiment, an anti-CD19 antibodyof the invention comprises the VH sequence 14H5 VH (SEQ ID NO:103) andthe VK sequence 14H5 VK (SEQ ID NO:111). In a specific embodiment, ananti-CD19 antibody of the invention comprises the VH sequence 14H5-YG VH(SEQ ID NO:107) and the VK sequence 14H5 VK (SEQ ID NO:111). In aspecific embodiment, an anti-CD19 antibody of the invention comprisesthe VH sequence 14H5-DG VH (SEQ ID NO:108) and the VK sequence 14H5 VK(SEQ ID NO:111). In a specific embodiment, an anti-CD19 antibody of theinvention comprises the VH sequence 14H5-LG VH (SEQ ID NO:109) and theVK sequence 14H5 VK (SEQ ID NO:111). In a specific embodiment, ananti-CD19 antibody of the invention comprises the VH sequence 14H5 VH(SEQ ID NO:103) and the VK sequence 16C9 VK (SEQ ID NO:113). In aspecific embodiment, an anti-CD19 antibody of the invention comprisesthe VH sequence 15D1 VH (SEQ ID NO:104) and the VK sequence 15D1 VK (SEQID NO:112). In a specific embodiment, an anti-CD19 antibody of theinvention comprises the VH sequence 15D7 VH (SEQ ID NO:105) and the VKsequence 14H5 VK (SEQ ID NO:111). In a specific embodiment, an anti-CD19antibody of the invention comprises the VH sequence 16C4 VH (SEQ IDNO:106) and the VK sequence 14H5 VK (SEQ ID NO:111). In a specificembodiment, an anti-CD19 antibody of the invention comprises the VHsequence 1A7 VH (SEQ ID NO:191) and the VK sequence 14H5 VK (SEQ IDNO:111). In a specific embodiment, an anti-CD19 antibody of theinvention comprises the VH sequence 1A7 VH (SEQ ID NO:191) and the VKsequence 3C3 VK (SEQ ID NO:193). In a specific embodiment, an anti-CD19antibody of the invention comprises the VH sequence 1A7 VH (SEQ IDNO:191) and the VK sequence 3E5 VK (SEQ ID NO:194). In a specificembodiment, an anti-CD19 antibody of the invention comprises the VHsequence 1A7 VH (SEQ ID NO:191) and the VK sequence 3D4 VK (SEQ IDNO:195). In a specific embodiment, an anti-CD19 antibody of theinvention comprises the VH sequence 1A7 VH (SEQ ID NO:191) and the VKsequence 5B5 VK (SEQ ID NO:197). In a specific embodiment, an anti-CD19antibody of the invention comprises the VH sequence 1A7 VH (SEQ IDNO:191) and the VK sequence 6F7 VK (SEQ ID NO:198). In a specificembodiment, an anti-CD19 antibody of the invention comprises the VHsequence 1A7 VH (SEQ ID NO:191) and the VK sequence 2D10 VK (SEQ IDNO:201). In a specific embodiment, an anti-CD19 antibody of theinvention comprises the VH sequence 1A7 VH (SEQ ID NO:191) and the VKsequence 5C11 VK (SEQ ID NO:202). In a specific embodiment, an anti-CD19antibody of the invention comprises the VH sequence 1A7 VH (SEQ IDNO:191) and the VK sequence 9G7 VK (SEQ ID NO:205). In a specificembodiment, an anti-CD19 antibody of the invention comprises the VHsequence 1A7 VH (SEQ ID NO:191) and the VK sequence 1H4 VK (SEQ IDNO:206). In a specific embodiment, an anti-CD19 antibody of theinvention comprises the VH sequence 1A7 VH (SEQ ID NO:191) and the VKsequence 5C4 VK (SEQ ID NO:207). In a specific embodiment, an anti-CD19antibody of the invention comprises the VH sequence 3B4 VH (SEQ IDNO:236) and the VK sequence 14H5 VK (SEQ ID NO:111). In a specificembodiment, an anti-CD19 antibody of the invention comprises the VHsequence 3F11 VH (SEQ ID NO:192) and the VK sequence 3F11 VK (SEQ IDNO:196). In a specific embodiment, an anti-CD19 antibody of theinvention comprises the VH sequence 16C4 VH (SEQ ID NO:106) and the VKsequence 1C11 VK (SEQ ID NO:199). In a specific embodiment, an anti-CD19antibody of the invention comprises the VH sequence 16C4 VH (SEQ IDNO:106) and the VK sequence 2B11 VK (SEQ ID NO:200). In a specificembodiment, an anti-CD19 antibody of the invention comprises the VHsequence 16C4 VH (SEQ ID NO:106) and the VK sequence 5D4 VK (SEQ IDNO:203). In a specific embodiment, an anti-CD19 antibody of theinvention comprises the VH sequence 16C4 VH (SEQ ID NO:106) and the VKsequence 6F7 VK (SEQ ID NO:198). In a specific embodiment, an anti-CD19antibody of the invention comprises the VH sequence 3F11 VH (SEQ IDNO:192) and the VK sequence 6C11 VK (SEQ ID NO:204). In a specificembodiment, an anti-CD19 antibody of the invention comprises anycombination of a VH and a VL listed in Table 1.

In certain embodiments, a light chain comprising a humanized VK of theinvention may be expressed with a heavy chain comprising a humanized VHof the invention to produce a humanized anti-CD19 antibody. In oneembodiment, a humanized anti-CD19 antibody described herein comprises aVK sequence selected from the group consisting of the sequencesdesignated HB12B-(A10/JK4) (SEQ ID NO:52); HB12B-364987 (or 364987) (SEQID NO:62); HB12B-3649 (or 3649) (SEQ ID NO:68); HB12B-36 (or 36) (SEQ IDNO:70), 7E12 VK (SEQ ID NO:110), 14H5 (SEQ ID NO:111), 15D1 (SEQ IDNO:112), 16C9 (SEQ ID NO:113), 3C3 (SEQ ID NO:193), 3E5 (SEQ ID NO:194),3D4 (SEQ ID NO:195), 3F11 (SEQ ID NO:196), 5B5 (SEQ ID NO:197), 6F7 (SEQID NO:198), 1C11 (SEQ ID NO:199), 2B11 (SEQ ID NO:200), 2D10 (SEQ IDNO:201), 5C11 (SEQ ID NO:202), 5D4 (SEQ ID NO:203), 6C11 (SEQ IDNO:204), 9G7 (SEQ ID NO:205), 1H4 (SEQ ID NO:206), AND 5C4 (SEQ IDNO:207). The aforementioned VK sequence may be paired with a VH sequencecomprising an amino acid sequence in its framework region selected fromthe group consisting of SEQ ID NO:36, 38, 40, and 42.

In certain embodiments, a heavy chain comprising a humanized VH of theinvention may be expressed with a light chain comprising a humanized VKof the invention to produce a humanized anti-CD19 antibody. In oneembodiment, a humanized anti-CD19 antibody described herein comprises aVH sequence selected from the group consisting of the sequencesdesignated HB12B-(3-72/JH4) (SEQ ID NO:34), 7E12 VH (SEQ ID NO:102),14H5 VH (SEQ ID NO:103), 15D1 VH (SEQ ID NO:104), 15D7 VH (SEQ IDNO:105), 16C4 VH (SEQ ID NO:106), 14H5-YG (SEQ ID NO:107), 14H5-DG (SEQID NO:108), 14H5-LG (SEQ ID NO:109), 1A7 (SEQ ID NO:191), 3C3 VH (SEQ IDNO:191), 6C11 VH (SEQ ID NO:191), 9G7 (SEQ ID NO:191), 3B4 VH (SEQ IDNO:236), and 3F11 VH (SEQ ID NO:192). The aforementioned VH sequence maybe paired with a VK sequence comprising an amino acid sequence in itsframework region selected from the group consisting of SEQ ID NO:54, SEQID NO:56, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:64, SEQ ID NO:58, SEQ IDNO:66, and SEQ ID NO:60.

In certain embodiments, a humanized VH or VK derived from the parentalHB12A or HB12B hybridoma may be expressed as a chimeric immunoglobulinlight chain or a chimeric immunoglobulin heavy chain to produce achimeric anti-CD19 antibody. In a particular embodiment, a humanized VHmay be expressed as a chimeric antibody comprising the HB12A VK (SEQ IDNO:4) or HB12B VK (SEQ ID NO:20). In another particular embodiment, ahumanized VK may be expressed as a chimeric antibody comprising theHB12A VH (SEQ ID NO:2) or HB12B VH (SEQ ID NO:18). In anotherembodiment, a chimeric anti-CD19 antibody may comprise the VK sequenceof HB12A VK (SEQ ID NO:4) or HB12B VK (SEQ ID NO:20) and may furthercomprise the VH sequence of HB12A VH (SEQ ID NO:2) or HB12B VH (SEQ IDNO:18).

In a particular embodiment, a humanized VH of the invention may furthercomprise a leader sequence of MGDNDIHFAFLSTGVHS (SEQ ID NO:83).

In another embodiment, a humanized VK of the invention may furthercomprise a leader sequence MDMRVPAQLLGLLLLWLPGAKC (SEQ ID NO:84)selected from the leader peptide of the human VKI-L12 gene.

Anti-CD19 antibodies described herein may have a high binding affinityfor the human CD19 (hCD19) antigen. For example, an antibody describedherein may have an association rate constant or k_(on) rate (antibody(Ab)+antigen (Ag)^(kon)→Ab-Ag) of at least 2×10⁵M⁻¹ s⁻¹, at least 5×10⁵M⁻¹ s¹ at least 10⁶ m⁻¹ s⁻¹, at least 5×10⁶ M⁻¹ s⁻¹ at least 10⁷M⁻¹ s⁻¹at least 5×10⁷ M⁻¹ s⁻¹, or at least 10⁸M⁻¹ s⁻¹.

In another embodiment, an anti-CD19 antibody of the invention may have ak_(off) rate ((Ab-Ag)^(koff)→antibody (Ab)+antigen (Ag)) of less than5×10⁻¹ s⁻¹, less than 10⁻¹ s⁻¹, less than 5×10⁻² s⁻¹, less than 10⁻²s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁻³ s⁻¹, less than 5×10⁻⁴ s⁻¹, orless than 10⁻⁴ s⁻¹. In a another embodiment, an antibody of theinvention has a k_(off) of less than 5×10⁻⁵ s⁻¹, less than 10⁻⁵ s⁻¹,less than 5×10⁻⁶ s⁻¹, less than 10⁻⁶ s⁻¹, less than 5×10⁻⁷ s⁻¹, lessthan 10⁻⁷ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁸ s⁻¹, less than5×10⁻⁹ s⁻¹, less than 10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In another embodiment, an anti-CD19 antibody of the invention may havean affinity constant or K_(a) (k_(on)/k_(off)) of at least 10² M⁻¹, atleast 5×10²M⁻¹, at least 10³M⁻¹, at least 5×10³ M⁻¹, at least 10⁴M⁻¹, atleast 5×10⁴M⁻¹, at least 10⁵ M⁻¹, at least 5×10⁵M⁻¹, at least 10⁶ M⁻¹,at least 5×10⁶ M⁻¹, at least 10⁷M⁻¹, at least 5×10⁷M⁻¹, at least 10⁸M⁻¹,at least 5×10⁸M⁻¹, at least 10⁹M⁻¹, at least 5×10⁹M⁻¹, at least 10¹⁰M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹M⁻¹ at least 10¹¹ M⁻¹, at least10¹² M⁻¹, at least 5×10¹² M⁻¹, at least 10¹³ M⁻¹ at least 5×10¹³ M⁻¹, atleast 10¹⁴ M⁻¹, at least 5×10¹⁴ M⁻¹, at least 10¹⁵ M⁻¹, or at least5×10¹⁵M⁻¹. In yet another embodiment, an anti-CD19 antibody of theinvention may have a dissociation constant or K_(d) (k_(off)/k_(on)) ofless than 5×10⁻² M, less than 10⁻²M, less than 5×10⁻³ M, less than10⁻³M, less than 5×10⁻⁴ M, less than 10⁻⁴ M, less than 5×10⁻⁵ M, lessthan 10⁻⁵M, less than 5×10⁻⁶ M, less than 10⁻⁶ M, less than 5×10⁻⁷ M,less than 10⁻⁷M, less than 5×10⁻⁸ M, less than 10⁻⁸M, less than 5×10⁻⁹M, less than 10⁻⁹M, less than 5×10⁻¹⁰ M, less than 10⁻¹⁰ M, less than5×10⁻¹¹M, less than 10⁻¹¹ M, less than 5×10⁻¹² M, less than 10⁻¹² M,less than 5×10⁻¹³ M, less than 10⁻¹³ M, less than 5×10⁻¹⁴ M, less than10⁻¹⁴ M, less than 5×10⁻¹⁵ M, or less than 10⁻¹⁵ M.

In one embodiment, an antibody of the invention used in accordance witha method described herein may immunospecifically bind to human CD19 andmay have a dissociation constant (K_(d)) of less than 3000 pM, less than2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, lessthan 750 pM, less than 500 pM, less than 250 pM, less than 200 pM, lessthan 150 pM, less than 100 pM, less than 75 pM as assessed using amethod described herein or known to one of skill in the art (e.g., aBIAcore assay, ELISA) (Biacore International AB, Uppsala, Sweden). In aspecific embodiment, an antibody of the invention used in accordancewith a method described herein may immunospecifically bind to a humanCD19 antigen and may have a dissociation constant (K_(d)) of between 25to 3400 pM, 25 to 3000 pM, 25 to 2500 pM, 25 to 2000 pM, 25 to 1500 pM,25 to 1000 pM, 25 to 750 pM, 25 to 500 pM, 25 to 250 pM, 25 to 100 pM,25 to 75 pM, 25 to 50 pM as assessed using a method described herein orknown to one of skill in the art (e.g., a BIAcore assay, ELISA). Inanother embodiment, an anti-CD19 antibody of the invention used inaccordance with a method described herein may immunospecifically bind tohCD19 and may have a dissociation constant (K_(d)) of 500 pM, 100 pM, 75pM or 50 pM as assessed using a method described herein or known to oneof skill in the art (e.g., a BIAcore assay, ELISA).

The invention further provides polynucleotides comprising a nucleotidesequence encoding a human, humanized, or chimeric anti-CD19 antibodydescribed herein or fragments thereof. The invention also encompassespolynucleotides that hybridize under stringent or lower stringencyhybridization conditions, e.g., as defined herein, to polynucleotidesthat encode a human, humanized, or chimeric antibody described hereinthat binds to hCD19.

Stringent hybridization conditions include, but are not limited to,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., highly stringent conditions such as hybridization tofilter-bound DNA in 6×SSC at about 45° C. followed by one or more washesin 0.1×SSC/0.2% SDS at about 60° C., or any other stringenthybridization conditions known to those skilled in the art (see, forexample, Ausubel, F. M. et al., eds. 1989 Current Protocols in MolecularBiology, vol. 1, Green Publishing Associates, Inc. and John Wiley andSons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

A polynucleotide encoding an antibody may also be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the immunoglobulinmay be chemically synthesized or obtained from a suitable source (e.g.,an antibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA+RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody) by PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

The present invention also provides polynucleotide sequences encoding VHand VK framework regions and CDRs of antibodies described herein as wellas expression vectors for their efficient expression in mammalian cells.

The present invention further provides for antibodies that mayefficiently deplete B cells expressing a recombinant human CD19 moleculein a hCD19 transgenic mouse model system (see, Yazawa et al., Proc NatlAcad Sci USA. 102(42):15178-83 (2005)). In a specific embodiment, ananti-CD-19 antibody of the invention may achieve B cell depletion thatis at least 20%, at least 30%, at least 40%, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%of the depletion achieved by the HB12B monoclonal antibody. In a furtherembodiment, an anti-CD19 antibody of the invention may achieve B celldepletion that is more complete than the depletion achieved by the HB12Bantibody. In one embodiment, an anti-CD19 antibody of the invention maydeplete circulating B cells, blood B cells, splenic B cells, marginalzone B cells, follicular B cells, peritoneal B cells, and/or bone marrowB cells. In a one embodiment, an anti-CD19 antibody of the invention mayachieve depletion of progenitor B cells, early pro-B cells, late pro-Bcells, large-pre-B cells, small pre-B cells, immature B cells, mature Bcells, antigen stimulated B cells, and/or plasma cells. In oneembodiment, B cell depletion by an anti-CD19 antibody of the inventionmay persist for at least 1 day, at least 2 days, at least 3 days, atleast 4 days, at least 5 days at least 6 days, at least 7 days, at least8 days, at least 9 days, at least 10 days, at least 15 days, at least 20days, at least 25 days, or at least 30 days. In another embodiment, Bcell depletion by an anti-CD19 antibody of the invention may persist forat least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks,at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks,at least 9 weeks, or at least 10 weeks. In a further embodiment, B celldepletion by an anti-CD19 antibody of the invention may persist for atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months, at least 7 months, at least 8months, at least 9 months, at least 10 months, at least 11 months or atleast 12 months.

The present invention also provides for antibodies that efficientlydeplete B cells in a human subject. In a specific embodiment, ananti-CD-19 antibody of the invention may achieve at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, or about 100% B cell depletion. In another embodiment,an anti-CD19 antibody of the invention may deplete B cell subsets in ahuman subject. In a specific embodiment, an anti-CD19 antibody of theinvention may deplete circulating B cells, blood B cells, splenic Bcells, marginal zone B cells, follicular B cells, peritoneal B cells,and/or bone marrow B cells. CD19 is present on the surface of B cells atall developmental stages. An anti-CD19 antibody may therefore deplete Bcells of all developmental stages. In a specific embodiment, ananti-CD19 antibody of the invention may achieve depletion of progenitorB cells, early pro-B cells, late pro-B cells, large-pre-B cells, smallpre-B cells, immature B cells, mature B cells, antigen stimulated Bcells, and/or plasma cells. Depletion of B cells may persist forextended periods of time. In one embodiment, B cell depletion by ananti-CD19 antibody of the invention may persist for at least 1 day, atleast 2 days, at least 3 days, at least 4 days, at least 5 days at least6 days, at least 7 days, at least 8 days, at least 9 days, at least 10days, at least 15 days, at least 20 days, at least 25 days, or at least30 days. In another embodiment, B cell depletion by an anti-CD19antibody of the invention may persist for at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least10 weeks. In a further embodiment, B cell depletion by an anti-CD19antibody of the invention may persist for at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, at least 7 months, at least 8 months, at least 9 months,at least 10 months, at least 11 months or at least 12 months.

In one embodiment, an anti-CD19 antibody of the invention depletes atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, or about 100% of circulating Bcells. In one embodiment, an anti-CD19 antibody of the inventiondepletes at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, or about 100% of blood Bcells. In one embodiment, an anti-CD19 antibody of the inventiondepletes at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, or about 100% of splenic Bcells. In one embodiment, an anti-CD19 antibody of the inventiondepletes at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, or about 100% of marginalzone B cells. In one embodiment, an anti-CD19 antibody of the inventiondepletes at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, or about 100% of follicularB cells. In one embodiment, an anti-CD19 antibody of the inventiondepletes at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, or about 100% of peritonealB cells. In one embodiment, an anti-CD19 antibody of the inventiondepletes at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, or about 100% of bonemarrow B cells. In one embodiment, an anti-CD19 antibody of theinvention depletes at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or about100% of progenitor B cells. In one embodiment, an anti-CD19 antibody ofthe invention depletes at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or about100% of early pro-B cells. In one embodiment, an anti-CD19 antibody ofthe invention depletes at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or about100% of late pro-B cells. In one embodiment, an anti-CD19 antibody ofthe invention depletes at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or about100% of large pre-B cells. In one embodiment, an anti-CD19 antibody ofthe invention depletes at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or about100% of small pre-B cells. In one embodiment, an anti-CD19 antibody ofthe invention depletes at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or about100% of immature B cells. In one embodiment, an anti-CD19 antibody ofthe invention depletes at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or about100% of mature B cells. In one embodiment, an anti-CD19 antibody of theinvention depletes at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or about100% of antigen stimulated B cells. In one embodiment, an anti-CD19antibody of the invention depletes at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, or about 100% of plasma cells. Depletion of B cells may persist forextended periods of time. In one embodiment, B cell depletion by ananti-CD19 antibody of the invention may persist for at least 1 day, atleast 2 days, at least 3 days, at least 4 days, at least 5 days at least6 days, at least 7 days, at least 8 days, at least 9 days, at least 10days, at least 15 days, at least 20 days, at least 25 days, or at least30 days. In another embodiment, B cell depletion by an anti-CD19antibody of the invention may persist for at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least10 weeks. In a further embodiment, B cell depletion by an anti-CD19antibody of the invention may persist for at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, at least 7 months, at least 8 months, at least 9 months,at least 10 months, at least 11 months or at least 12 months.

B cell malignancies are characterized by the pathological expansion ofspecific B cell subsets, for example, precursor B cell acutelymphoblastic leukemia is characterized by an abnormal expansion of Bcells corresponding to pro-B cell/Pre-B cell developmental stages. Themalignant B cells maintain cell surface expression of normal B cellmarkers such as CD19. An anti-CD19 antibody may therefore depletemalignant B cells in a human subject. In a specific embodiment, ananti-CD19 antibody of the invention may achieve at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 100% depletion of malignantB cells in a human subject.

In one embodiment, a humanized anti-CD19 antibody described hereinmediates antibody-dependent cellular cytotoxicity (ADCC),complement-dependent cell-mediated cytotoxicity (CDC), and/or apoptosis.In one embodiment, a humanized anti-CD19 antibody of the inventionmediates antibody-dependent cellular cytotoxicity (ADCC) and/orapoptosis. In one embodiment, an anti-CD19 antibody of the invention hasenhanced antibody-dependent cellular cytotoxicity (ADCC). In oneembodiment, an anti-CD19 antibody of the invention comprises a variantFc region that mediates enhanced antibody-dependent cellularcytotoxicity (ADCC). In one embodiment, an anti-CD19 antibody of theinvention comprises an Fc region having complex N-glycoside-linked sugarchains linked to Asn297 in which fucose is not bound toN-acetylglucosamine in the reducing end, wherein said Fc region mediatesenhanced antibody-dependent cellular cytotoxicity (ADCC).

The present invention further provides for anti-CD19 antibodies that mayefficiently inhibit in vitro stimulated B cell proliferation.Proliferation of isolated peripheral B cells may be induced by variousstimuli, for example, but not limited to stimulation by anti-IgMantibody, CD40 or CpG. These stimuli may be delivered in alone or incombination with each other.

In one embodiment, an anti-CD19 antibody of the invention inhibits invitro stimulated B cell proliferation. In a another embodiment, ananti-CD19 antibody described herein inhibits in vitro B cellproliferation induced by anti-IgM/CpG or anti-IgM/CD40 stimulation. Inone embodiment, an anti-CD19 antibody of the invention inhibits in vitrostimulated B cell proliferation by at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50% or atleast about 75%.

In one embodiment, an Fc variant anti-CD19 antibody of the inventioninhibits in vitro B cell proliferation induced by anti-IgM/CpG oranti-IgM/CD40 stimulation, wherein said Fc variant has altered bindingaffinity to one or more Fc ligand relative to a comparable non-variantmolecule. In a specific embodiment, an Fc variant anti-CD19 antibody ofthe invention inhibits in vitro B cell proliferation induced byanti-IgM/CpG or anti-IgM/CD40 stimulation, wherein said Fc variant hasenhanced binding to Fc gamma receptor IIB relative to a comparablenon-variant Fc domain. In a further specific embodiment, an Fc variantanti-CD19 antibody of the invention inhibits in vitro stimulated B cellproliferation by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50% or at least about 75%. Inanother embodiment, an Fc variant anti-CD19 antibody of the inventioninhibits in vitro stimulated B cell proliferation, wherein said variantFc domain has an affinity for Fc gamma receptor IIB that is at least 2fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold greater than that of a comparable non-variant Fc domain.

The present invention also relates to a method of treating a B cellmalignancy in a human comprising administering to a human in needthereof, a human, humanized or chimeric anti-CD19 antibody that maymediate human antibody-dependent cellular cytotoxicity (ADCC),complement-dependent cell-mediated cytotoxicity (CDC), and/or apoptosisin an amount sufficient to deplete circulating B cells. In a particularaspect, the present invention also concerns methods of treating a B cellmalignancy in a human comprising administration of a therapeuticallyeffective regimen of a human, humanized, or chimeric anti-CD19 antibody,which is of the IgG1 or IgG3 human isotype.

The present invention further relates to a method of treating anautoimmune disease or disorder in a human comprising administering to ahuman in need thereof a human, humanized, or chimeric anti-CD19 antibodythat may mediate human ADCC, CDC, and/or apoptosis in an amountsufficient to deplete circulating B cells. The present invention alsoconcerns methods of treating autoimmune disorders comprisingadministration of a therapeutically effective regimen of a human,humanized, or chimeric anti-CD19 antibody which is of the IgG1 or IgG3human isotype.

The present invention also provides methods for treating or preventinghumoral rejection in a human transplant recipient in need thereofcomprising administering to the recipient a human, humanized, orchimeric anti-CD19 antibody of the invention in an amount that maydeplete circulating B cells, or circulating immunoglobulin, or both. Inother embodiments, the invention provides methods for preventing graftrejection or graft versus host disease in a human transplant recipientin need thereof comprising contacting a graft prior to transplantationwith an amount of a human, humanized, or chimeric anti-CD19 antibodythat may deplete B cells from the graft.

5.2. Production of Humanized Anti-CD19 Antibodies

Humanized antibodies described herein can be produced using a variety oftechniques known in the art, including, but not limited to, CDR-grafting(see e.g., European Patent No. EP 239,400; International Publication No.WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089,each of which is incorporated herein in its entirety by reference),veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 andEP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498;Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguskaet al., 1994, Proc. Natl. Acad. Sci., 91:969-973, each of which isincorporated herein by its entirety by reference), chain shuffling (see,e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in itsentirety by reference), and techniques disclosed in, e.g., U.S. Pat. No.6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al.,Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79(2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska etal., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22(1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J.Mol. Biol., 235(3):959-73 (1994), each of which is incorporated hereinin its entirety by reference. Often, FW residues in the FW regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These FWsubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and FW residues to identify FWresidues important for antigen binding and sequence comparison toidentify unusual FW residues at particular positions. (See, e.g., Queenet al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties.)

A humanized anti-CD19 antibody has one or more amino acid residuesintroduced into it from a source which is nonhuman. These nonhuman aminoacid residues are often referred to as “import” residues, which aretypically taken from an “import” variable domain. Thus, humanizedantibodies comprise one or more CDRs from nonhuman immunoglobulinmolecules and framework regions from human. Humanization of antibodiesis well-known in the art and can essentially be performed following themethod of Winter and co-workers (Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody, i.e.,CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Pat.Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640,the contents of which are incorporated by reference herein in theirentirety). In such humanized chimeric antibodies, substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a nonhuman species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FW residues are substituted by residues from analogoussites in rodent antibodies. Humanization of anti-CD19 antibodies canalso be achieved by veneering or resurfacing (EP 592,106; EP 519,596;Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al.,Protein Engineering, 7(6):805-814 (1994); and Roguska et al., Proc.Natl. Acad. Sci., 91:969-973 (1994)) or chain shuffling (U.S. Pat. No.5,565,332), the contents of which are incorporated herein by referencein their entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequences which are mostclosely related to that of the rodent are then screened for thepresences of specific residues that may be critical for antigen binding,appropriate structural formation and/or stability of the intendedhumanized mAb (Sims et al., J. Immunol., 151:2296 (1993); Chothia etal., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference in their entirety). The resulting FWsequences matching the desired criteria are then be used as the humandonor FW regions for the humanized antibody.

Another method uses a particular FW derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same FW may be used for several different humanizedanti-CD19 antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993), the contents ofwhich are incorporated herein by reference in their entirety).

Anti-CD19 antibodies can be humanized with retention of high affinityfor CD19 and other favorable biological properties. According to oneaspect of the invention, humanized antibodies are prepared by a processof analysis of the parental sequences and various conceptual humanizedproducts using three-dimensional models of the parental and humanizedsequences. Three-dimensional immunoglobulin models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind CD19. In this way,FW residues can be selected and combined from the recipient and importsequences so that the desired antibody characteristic, for exampleaffinity for CD19, is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

A “humanized” antibody may retain a similar antigenic specificity as theoriginal antibody, i.e., in the present invention, the ability to bindhuman CD19 antigen. However, using certain methods of humanization, theaffinity and/or specificity of binding of the antibody for human CD19antigen may be altered using methods of “directed evolution,” asdescribed by Wu et al., J. Mol. Biol, 294:151 (1999), the contents ofwhich are incorporated herein by reference herein in their entirety.

Humanized anti-CD19 antibodies described herein can be constructed bythe selection of distinct human framework regions for grafting of theHB12A or HB12B complementarity determining regions, or “CDR's” asdescribed in the sections that follow. The invention encompasses anumber of humanized versions of the mouse HB12A and HB12B antibody aswell as chimeric versions, designated chHB12A and chHB12B.

5.3. Monoclonal Anti-CD19 Antibodies

A monoclonal anti-CD19 antibody exhibits binding specificity to humanCD19 antigen and may mediate human ADCC, CDC and/or apoptoticmechanisms. Such an antibody can be generated using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. Antibodies arehighly specific, being directed against a single antigenic site. Anengineered anti-CD19 antibody can be produced by any means known in theart, including, but not limited to, those techniques described below andimprovements to those techniques. Large-scale high—yield productiontypically involves culturing a host cell that produces the engineeredanti-CD19 antibody and recovering the anti-CD19 antibody from the hostcell culture.

5.3.1. Hybridoma Technique

Monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling et al., in Monoclonal Antibodies and TCell Hybridomas, 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated herein by reference in their entireties). For example, inthe hybridoma method, a mouse or other appropriate host animal, such asa hamster or macaque monkey, is immunized to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Lymphocytes may also beimmunized in vitro. Lymphocytes then are fused with myeloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that contains one or more substances that inhibit thegrowth or survival of the unfuscd, parental mycloma cells. For example,if the parental mycloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asthose derived from MOPC-21 and MPC-11 mouse tumors available from theSalk Institute Cell Distribution Center, San Diego, Calif., USA, andSP-2 or X63-Ag8.653 cells available from the American Type CultureCollection, Rockville, Md., USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the human CD19antigen. The binding specificity of monoclonal antibodies produced byhybridoma cells can be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI 1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

5.3.2. Recombinant DNA Techniques

DNA encoding an anti-CD19 antibody described herein is readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of anti-CD19 antibodies). Thehybridoma cells serve as a source of such DNA. Once isolated, the DNAmay be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of anti-CD19 antibodiesin the recombinant host cells.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of affected tissues). The DNA encoding the VH and VL domainsare recombined together with an scFv linker by PCR and cloned into aphagemid vector. The vector is electroporated in E. coli and the E. coliis infected with helper phage. Phage used in these methods is typicallyfilamentous phage including fd and M13 and the Vii and VL domains areusually recombinantly fused to either the phage gene III or gene VIII.Phage expressing an antigen-binding domain that binds to a particularantigen can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., 1995, J. Immunol. Methods, 182:41-50; Ames et al., 1995, J.Immunol. Methods, 184:177-186; Kettleborough et al., 1994, Eur. J.Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18; Burton etal., 1994, Advances in Immunology, 57:191-280; International ApplicationNo. PCT/GB91/O1 134; International Publication Nos. WO 90/02809, WO91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409,5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698,5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen-binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)2 fragments can also be employed using methods knownin the art such as those disclosed in PCT Publication No. WO 92/22324;Mullinax et al., 1992, BioTechniques, 12(6):864-869; Sawai et al., 1995,AJRI, 34:26-34; and Better et al., 1988, Science, 240:1041-1043 (saidreferences incorporated by reference in their entireties).

Antibodies may be isolated from antibody phage libraries generated usingthe techniques described in McCafferty et al., Nature, 348:552-554(1990). Clackson et al., Nature, 352:624-628 (1991). Marks et al., J.Mol. Biol., 222:581-597 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Chain shufflingcan be used in the production of high affinity (nM range) humanantibodies (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of anti-CD19 antibodies.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. The vectors for expressing the VH or VL domains may comprise anEF-1a promoter, a secretion signal, a cloning site for the variabledomain, constant domains, and a selection marker such as neomycin. TheVH and VL domains may also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

5.4. Chimeric Antibodies

The anti-CD19 antibodies herein specifically include chimeric antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while another portion of the chain(s) is identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a nonhuman primate (e.g.,Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and humanconstant region sequences (U.S. Pat. No. 5,693,780).

5.5. Altered/Mutant Antibodies

Anti-CD19 antibodies of compositions and methods described herein can bemutant antibodies. As used herein, “antibody mutant” or “alteredantibody” refers to an amino acid sequence variant of an anti-CD19antibody wherein one or more of the amino acid residues of an anti-CD19antibody have been modified. The modifications to the amino acidsequence of an anti-CD19 antibody include modifications to the sequencethat may improve affinity or avidity of the antibody for its antigen,and/or modifications to the Fc portion of the antibody that may improveeffector function.

The present invention therefore relates to human, humanized, andchimeric anti-CD19 antibodies disclosed herein as well as altered/mutantderivatives thereof including, but not limited to ones exhibitingaltered human CD19 binding characteristics; e.g. altered associationconstants k_(ON), dissociation constants k_(OFF), and/or equilibriumconstant or binding affinity, K_(D). In certain embodiments the K_(D) ofan anti-CD19 antibody described herein, or an altered/mutant derivativethereof, for human CD19 may be no more than about 10⁶M, 10⁷M, 10⁻⁸M, or10⁻⁹M. Methods and reagents suitable for determination of such bindingcharacteristics of an antibody of the present invention, or analtered/mutant derivative thereof, are known in the art and/or arecommercially available (se above and, e.g., U.S. Pat. No. 6,849,425,U.S. Pat. No. 6,632,926, U.S. Pat. No. 6,294,391, and U.S. Pat. No.6,143,574, each of which is hereby incorporated by reference in itsentirety). Moreover, equipment and software designed for such kineticanalyses are commercially available (e.g. Biacore® A100, and Biacore®2000 instruments; Biacore International AB, Uppsala, Sweden).

The modifications may be made to any known anti-CD19 antibodies oranti-CD19 antibodies identified as described herein. Such alteredantibodies necessarily have less than 100% sequence identity orsimilarity with a known anti-CD19 antibody. By way of example, analtered antibody may have an amino acid sequence that is within therange of from about 25% to about 95% identical or similar to the aminoacid sequence of either the heavy or light chain variable domain of ananti-CD19 antibody as described herein. An altered antibody may have anamino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%,85%, 90%, or 95% amino acid sequence identity or similarity with theamino acid sequence of either the heavy or light chain variable domainof an anti-CD19 antibody as described herein. In another embodiment, analtered antibody may have an amino acid sequence having at least 25%,35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequenceidentity or similarity with the amino acid sequence of the heavy chainCDR1, CDR2, or CDR3 of an anti-CD19 antibody as described herein. In oneembodiment, an altered antibody may maintain human CD19 bindingcapability. In certain embodiments, an anti-CD19 antibody as describedherein may comprise a VH that is at least or about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore identical to the amino acid sequence of HB12B-(3-72/JH4) (SEQ IDNO:34), HB12A VH (SEQ ID NO:2) HB12B VH (SEQ ID NO:18), 7E12 VH (SEQ IDNO:102), 14H5 VH (SEQ ID NO:103), 15D1 VH (SEQ ID NO:104), 15D7 VH (SEQID NO:105), 16C4 VH (SEQ ID NO:106), 14H5-YG (SEQ ID NO:107), 14H5-DG(SEQ ID NO:108), 14H5-LG (SEQ ID NO:109), 1A7 VH, 3C3 VH, 3E5 VH, 3D4VH, 9G7 VH (SEQ ID NO:191), 3B4 VH (SEQ ID NO: 236), 3F11 VH or 6C11 VH(SEQ ID NO:192). In certain embodiments, an anti-CD19 antibody asdescribed herein may comprise a VH that is at least or about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or more identical to the amino acid sequence of any of the VHdomains, VL domains, or CDRs listed in Table 1.

In another embodiment, an altered antibody may have an amino acidsequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or95% amino acid sequence identity or similarity with the amino acidsequence of FW1, FW2, FW3, or FW4 regions of HB12B-(3-72/JH4) (SEQ IDNO:34), HB12A VH (SEQ ID NO:2) HB12B VH (SEQ ID NO:18), 7E12 VH (SEQ IDNO:102), 14H5 VH (SEQ ID NO:103), 15D1 VH (SEQ ID NO:104), 15D7 VH (SEQID NO:105), 16C4 VH (SEQ ID NO:106), 14H5-YG (SEQ ID NO:107), 14H5-DG(SEQ ID NO:108), 14H5-LG (SEQ ID NO:109), 1A7 VH, 3C3 VH, 3E5 VH, 3D4VH, 9G7 VH (SEQ ID NO:191), 3B4 VH (SEQ ID NO: 236), 3F11 VH or 6C11 VH(SEQ ID NO:192). In another embodiment, an altered antibody may have anamino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%,85%, 90%, or 95% amino acid sequence identity or similarity with theamino acid sequence of FW1, FW2, FW3, or FW4 regions of any of the VH orVL domains listed in Table 1.

In another embodiment, an altered antibody may have an amino acidsequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or95% amino acid sequence identity or similarity with the amino acidsequence of the light chain CDR1, CDR2, or CDR3 of an anti-CD19 antibodyas described herein. In certain embodiments, an anti-CD19 antibody ofthe invention may comprise a VL that is at least or about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or more identical to an amino acid sequence of HB12A VK (SEQ IDNO:4), HB12B VK (SEQ ID NO:20), HB12B-(A10-Jk4) (SEQ ID NO:52),HB12B-364987 (or 364987) (SEQ ID NO:62), HB12B-3649 (or 3649) (SEQ IDNO:68), HB12B-36 (or 36) (SEQ ID NO:70), 7E12 VK (SEQ ID NO:110), 14H5(SEQ ID NO:111), 15D1 (SEQ ID NO:112), 16C9 (SEQ ID NO:113), 3C3 VK (SEQID NO:193), 3E5 VK (SEQ ID NO:194), 3D4 VK (SEQ ID NO:195), 3F1 VK (SEQID NO:196), 5B5 VK (SEQ ID NO:197), 6F7 VK (SEQ ID NO:198), 1C11 VK (SEQID NO:199), 2B11 VK (SEQ ID NO:200), 2D10 VK (SEQ ID NO:201), 5C11 VK(SEQ ID NO:202), 5D4 VK (SEQ ID NO:203), 6C11 VK (SEQ ID NO:204), 9G7 VK(SEQ ID NO:205), 1H4 VK (SEQ ID NO:206), or 5C4 VK (SEQ ID NO:207).

In another embodiment, an altered antibody may have an amino acidsequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or95% amino acid sequence identity or similarity with the amino acidsequence of FW1, FW2, FW3, or FW4 regions of HB12A VK (SEQ ID NO:4),HB12B VK (SEQ ID NO:20), HB12B-(A10-Jk4) (SEQ ID NO:52), HB12B-364987(or 364987) (SEQ ID NO:62), HB12B-3649 (or 3649) (SEQ ID NO:68),HB12B-36 (or 36) (SEQ TD NO:70), 7E12 VK (SEQ ID NO:110), 14H5 (SEQ IDNO:111), 15D1 (SEQ ID NO:112), 16C9 (SEQ ID NO:113), 3C3 VK (SEQ IDNO:193), 3E5 VK (SEQ ID NO:194), 3D4 VK (SEQ ID NO:195), 3F1 VK (SEQ IDNO:196), 5B5 VK (SEQ ID NO:197), 6F7 VK (SEQ ID NO:198), 1C11 VK (SEQ IDNO:199), 2B11 VK (SEQ ID NO:200), 2D10 VK (SEQ ID NO:201), 5C11 VK (SEQID NO:202), 5D4 VK (SEQ ID NO:203), 6C11 VK (SEQ ID NO:204), 9G7 VK (SEQID NO:205), 1H4 VK (SEQ ID NO:206), or 5C4 VK (SEQ ID NO:207).

Identity or similarity with respect to a sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) or similar (i.e., amino acid residue fromthe same group based on common side-chain properties, see below) withanti-CD19 antibody residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence outside of thevariable domain shall be construed as affecting sequence identity orsimilarity.

“% identity,” as known in the art, is a measure of the relationshipbetween two polynucleotides or two polypeptides, as determined bycomparing their sequences. In general, the two sequences to be comparedare aligned to give a maximum correlation between the sequences. Thealignment of the two sequences is examined and the number of positionsgiving an exact amino acid or nucleotide correspondence between the twosequences determined, divided by the total length of the alignment andmultiplied by 100 to give a % identity figure. This % identity figuremay be determined over the whole length of the sequences to be compared,which is particularly suitable for sequences of the same or very similarlength and which are highly homologous, or over shorter defined lengths,which is more suitable for sequences of unequal length or which have alower level of homology.

For example, sequences can be aligned with the software clustalw underUnix which generates a file with an “.aln” extension, this file can thenbe imported into the Bioedit program (Hall, T. A. 1999, BioEdit: ause/friendly biological sequence alignment editor and analysis programfor Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98) which opens the.aln file. In the Bioedit window, one can choose individual sequences(two at a time) and alignment them. This method allows for comparison ofthe entire sequence.

Methods for comparing the identity of two or more sequences are wellknown in the art. Thus for instance, programs are available in theWisconsin Sequence Analysis Package, version 9.1 (Devereux J. et al.,Nucleic Acids Res., 12:387-395, 1984, available from Genetics ComputerGroup, Madison, Wis., USA). The determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the programs BESTFIT and GAP, may be used todetermine the % identity between two polynucleotides and the % identitybetween two polypeptide sequences. BESTFIT uses the “local homology”algorithm of Smith and Wateiinan (Advances in Applied Mathematics,2:482-489, 1981) and finds the best single region of similarity betweentwo sequences. BESTFIT is more suited to comparing two polynucleotide ortwo polypeptide sequences which are dissimilar in length, the programassuming that the shorter sequence represents a portion of the longer.In comparison, GAP aligns two sequences finding a “maximum similarity”according to the algorithm of Neddleman and Wunsch (J. Mol. Biol.,48:443-354, 1970). GAP is more suited to comparing sequences which areapproximately the same length and an alignment is expected over theentire length. Preferably the parameters “Gap Weight” and “LengthWeight” used in each program are 50 and 3 for polynucleotides and 12 and4 for polypeptides, respectively. Preferably % identities andsimilarities are determined when the two sequences being compared areoptimally aligned.

Other programs for determining identity and/or similarity betweensequences are also known in the art, for instance the BLAST family ofprograms (Karlin & Altschul, 1990, Proc. Natl. Acad. Sci. USA,87:2264-2268, modified as in Karlin & Altschul, 1993, Proc. Natl. Acad.Sci. USA, 90:5873-5877, available from the National Center forBiotechnology Information (NCB), Bethesda, Md., USA and accessiblethrough the home page of the NCBI). These programs are non-limitingexamples of a mathematical algorithm utilized for the comparison of twosequences. Such an algorithm is incorporated into the NBLAST and XBLASTprograms of Altschul et al., 1990, J. Mol. Biol., 215:403-410. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecule encoding all or a portion if an anti-CD19 antibody of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecule of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402.PSI-Blast can also be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

Another non-limiting example of a program for determining identityand/or similarity between sequences known in the art is FASTA (PearsonW. R. and Lipman D. J., Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988,available as part of the Wisconsin Sequence Analysis Package).Preferably the BLOSUM62 amino acid substitution matrix (Henikoff S. andHenikoff J. G., Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) isused in polypeptide sequence comparisons including where nucleotidesequences are first translated into amino acid sequences beforecomparison.

Yet another non-limiting example of a program known in the art fordetermining identity and/or similarity between amino acid sequences isSeqWeb Software (a web-based interface to the GCG Wisconsin Package: Gapprogram) which is utilized with the default algorithm and parametersettings of the program: blosum62, gap weight 8, length weight 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

Preferably the program BESTFIT is used to determine the % identity of aquery polynucleotide or a polypeptide sequence with respect to apolynucleotide or a polypeptide sequence of the present invention, thequery and the reference sequence being optimally aligned and theparameters of the program set at the default value.

To generate an altered antibody, one or more amino acid alterations(e.g., substitutions) are introduced in one or more of the hypervariableregions of the species-dependent antibody. One or more alterations(e.g., substitutions) of framework region residues may also beintroduced in an anti-CD19 antibody where these result in an improvementin the binding affinity of the antibody mutant for the antigen from thesecond mammalian species. Examples of framework region residues tomodify include those which non-covalently bind antigen directly (Amit etal., Science, 233:747-753 (1986)); interact with/effect the conformationof a CDR (Chothia et al., J. Mol. Biol., 196:901-917 (1987)); and/orparticipate in the V_(L)-V_(H) interface (EP 239 400B1). In certainembodiments, modification of one or more of such framework regionresidues results in an enhancement of the binding affinity of theantibody for the antigen from the second mammalian species. For example,from about one to about five framework residues may be altered in thisembodiment of the invention. Sometimes, this may be sufficient to yieldan antibody mutant suitable for use in preclinical trials, even wherenone of the hypervariable region residues have been altered. Normally,however, an altered antibody will comprise additional hypervariableregion alteration(s).

The hypervariable region residues which are altered may be changedrandomly, especially where the starting binding affinity of an anti-CD19antibody for the antigen from the second mammalian species is such thatsuch randomly produced altered antibody can be readily screened.

One useful procedure for generating such an altered antibody is called“alanine scanning mutagenesis” (Cunningham and Wells, Science,244:1081-1085 (1989)). Here, one or more of the hypervariable regionresidue(s) are replaced by alanine or polyalanine residue(s) to affectthe interaction of the amino acids with the antigen from the secondmammalian species. Those hypervariable region residue(s) demonstratingfunctional sensitivity to the substitutions then are refined byintroducing additional or other mutations at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. The Ala-mutants produced this way arescreened for their biological activity as described herein.

Another procedure for generating such an altered antibody involvesaffinity maturation using phage display (Hawkins et al., J. Mol. Biol.,254:889-896 (1992) and Lowman et al., Biochemistry, 30(45):10832-10837(1991)). Briefly, several hypervariable region sites (e.g., 6-7 sites)are mutated to generate all possible amino acid substitutions at eachsite. The antibody mutants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene 111product of M13 packaged within each particle. The phage-displayedmutants are then screened for their biological activity (e.g., bindingaffinity) as herein disclosed.

Mutations in antibody sequences may include substitutions, deletions,including internal deletions, additions, including additions yieldingfusion proteins, or conservative substitutions of amino acid residueswithin and/or adjacent to the amino acid sequence, but that result in a“silent” change, in that the change produces a functionally equivalentanti-CD19 antibody. Conservative amino acid substitutions may be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. In addition, glycine and proline are residues that caninfluence chain orientation. Non-conservative substitutions will entailexchanging a member of one of these classes for a member of anotherclass. Furthermore, if desired, non-classical amino acids or chemicalamino acid analogs can be introduced as a substitution or addition intothe antibody sequence. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl aminoacids, and amino acid analogs in general.

In another embodiment, the sites selected for modification are affinitymatured using phage display (see above).

Any technique for mutagenesis known in the art can be used to modifyindividual nucleotides in a DNA sequence, for purposes of making aminoacid substitution(s) in the antibody sequence, or for creating/deletingrestriction sites to facilitate further manipulations. Such techniquesinclude, but are not limited to, chemical mutagenesis, in vitrosite-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA, 82:488(1985); Hutchinson, C. et al., J. Biol. Chem., 253:6551 (1978)),oligonucleotide-directed mutagenesis (Smith, Ann. Rev. Genet.,19:423-463 (1985); Hill et al., Methods Enzymol., 155:558-568 (1987)),PCR-based overlap extension (Ho et al., Gene, 77:51-59 (1989)),PCR-based megaprimer mutagenesis (Sarkar et al., Biotechniques,8:404-407 (1990)), etc. Modifications can be confirmed bydouble-stranded dideoxy DNA sequencing.

In certain embodiments of the invention, an anti-CD19 antibody can bemodified to produce fusion proteins; i.e., the antibody, or a fragmentthereof, fused to a heterologous protein, polypeptide or peptide. Incertain embodiments, the protein fused to the portion of an anti-CD19antibody is an enzyme component of Antibody-Directed Enzyme ProdrugTherapy (ADEPT). Examples of other proteins or polypeptides that can beengineered as a fusion protein with an anti-CD19 antibody include, butare not limited to toxins such as ricin, abrin, ribonuclease, DNase I,Staphylococcal enterotoxin-A, pokeweed anti-viral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See,for example, Pastan et al., Cell, 47:641 (1986), and Goldenberg et al.,Cancer Journal for Clinicians, 44:43 (1994). Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,non-binding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, for example, WO 93/21232 published Oct. 28, 1993.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of the antiCD19 antibody or fragmentsthereof (e.g., an antibody or a fragment thereof with higher affinitiesand lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997,Curr. Opinion Biotechnol., 8:724-33; Harayama, 1998, Trends Biotechnol.16(2):76-82; Hansson et al., 1999, J Mol. Biol., 287:265-76; and Lorenzoand Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents andpublications are hereby incorporated by reference in its entirety). Theantibody can further be a binding-domain immunoglobulin fusion proteinas described in U.S. Publication 20030118592, U.S. Publication200330133939, and PCT Publication WO 02/056910, all to Ledbetter et al.,which are incorporated herein by reference in their entireties.

5.6. Domain Antibodies

Anti-CD19 antibodies of compositions and methods of the invention can bedomain antibodies, e.g., antibodies containing the small functionalbinding units of antibodies, corresponding to the variable regions ofthe heavy (VH) or light (VL) chains of human antibodies. Examples ofdomain antibodies include, but are not limited to, those available fromDomantis Limited (Cambridge, UK) and Domantis Inc. (Cambridge, Mass.,USA) that are specific to therapeutic targets (see, for example,WO04/058821; WO04/003019; U.S. Pat. Nos. 6,291,158; 6,582,915;6,696,245; and 6,593,081). Commercially available libraries of domainantibodies can be used to identify anti-CD19 domain antibodies. Incertain embodiments, anti-CD19 antibodies comprise a CD19 functionalbinding unit and a Fc gamma receptor functional binding unit.

In one embodiment, an anti-CD19 domain antibody may comprise any one of,or any combination of the CDRs of the heavy or light chains of the HB12Aor HB12B monoclonal antibodies.

In another embodiment, an anti-CD19 domain antibody may comprise CDR3 ofHB12A or HB12B VHs together with any combination of the CDRs comprisedby the heavy or light chains variable regions of the HB12A or HB12Bmonoclonal antibodies. An anti-CD19 domain antibody may also compriseCDR3 of HB12A or HB12B VKs together with any combination of the CDRscomprised by the heavy or light chains variable regions of the HB12A orHB12B monoclonal antibodies.

In yet another embodiment, an anti-CD19 domain antibody may compriseCDR3 of HB12A or HB12B VHs. An anti-CD19 domain antibody may alsocomprise CDR3 of HB12A or HB12B VKs.

5.7. Diabodies

In certain embodiments of the invention, anti-CD19 antibodies are“diabodies”. The term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

5.8. Vaccibodies

In certain embodiments of the invention, anti-CD19 antibodies areVaccibodies. Vaccibodies are dimeric polypeptides. Each monomer of avaccibody consists of a scFv with specificity for a surface molecule onAPC connected through a hinge region and a Cγ3 domain to a second scFv.In other embodiments of the invention, vaccibodies containing as one ofthe scFv's an anti-CD19 antibody fragment may be used to juxtapose thoseB cells to be destroyed and an effector cell that mediates ADCC. Forexample, see, Bogen et al., U.S. Patent Application Publication No.20040253238.

5.9. Linear Antibodies

In certain embodiments of the invention, anti-CD19 antibodies are linearantibodies. Linear antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H1)-V_(H)-C_(H1)) which form a pair of antigen-bindingregions. Linear antibodies can be bispecific or monospecific. See,Zapata et al., Protein Eng., 8(10):1057-1062 (1995).

5.10. Parent Antibody+

In certain embodiments of the invention, anti-CD19 antibody is a parentantibody. A “parent antibody” is an antibody comprising an amino acidsequence which may lack, or may be deficient in, one or more amino acidresidues in or adjacent to one or more hypervariable regions thereofcompared to an altered/mutant antibody as herein disclosed. Thus, theparent antibody may have a shorter hypervariable region than thecorresponding hypervariable region of an antibody mutant as hereindisclosed. The parent polypeptide may comprise a native antibodysequence (i.e., a naturally occurring, including a naturally occurringallelic variant) or an antibody sequence with pre-existing amino acidsequence modifications (such as other insertions, deletions and/orsubstitutions) of a naturally occurring sequence. The parent antibodymay be a humanized antibody or a human antibody.

5.11. Antibody Fragments

“Antibody fragments” comprise a portion of a full-length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods, 24:107-117 (1992) and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above. Fab′-SHfragments can also be directly recovered from E. coli and chemicallycoupled to form F(ab′)₂ fragments (Carter et al., Bio/Technology,10:163-167 (1992)). According to another approach, F(ab′)₂ fragments canbe isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner. In other embodiments, the antibody of choiceis a single-chain Fv fragment (scFv). See, for example, WO 93/16185. Incertain embodiments, the antibody is not a Fab fragment.

5.12. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the B cell surface marker. Other suchantibodies may bind a first B cell marker and further bind a second Bcell surface marker. An anti-B cell marker binding arm may also becombined with an arm which binds to a triggering molecule on a leukocytesuch as a T cell receptor molecule (e.g., CD2 or CD3), or Fc receptorsfor IgG (FcγR), so as to focus cellular defense mechanisms to the Bcell. Bispecific antibodies may also be used to localize cytotoxicagents to the B cell. These antibodies possess a B cell marker-bindingarm and an arm which binds the cytotoxic agent (e.g., saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methola-exate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull-length antibodies or antibody fragments (e.g., F(ab′): bispecificantibodies).

Methods for making bispecific antibodies are known in the art. (See, forexample, Millstein et al., Nature, 305:537-539 (1983); Traunecker etal., EMBO 1, 10:3655-3659 (1991); Suresh et al., Methods in Enzymology,121:210 (1986); Kostelny et al., J. Immunol., 148(5):1547-1553 (1992);Hollinger et al., Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993); Gruberet al., J. Immunol., 152:5368 (1994); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,81; 95,731,168; 4,676,980; and4,676,980, WO 94/04690; WO 91/00360; WO 92/200373; WO 93/17715; WO92/08802; and EP 03089.)

In one embodiment, where an anti-CD19 antibody of compositions andmethods of the invention is bispecific, the anti-CD19 antibody may behuman or humanized and may have specificity for human CD19 and anepitope on a T cell or may be capable of binding to a human effectorcell such as, for example, a monocyte/macrophage and/or a natural killercell to effect cell death.

In one embodiment, an anti-CD19 antibody of the invention is abispecific antibody capable of specifically binding to a first andsecond antigen, wherein said first antigen is human CD19 and said secondantigen is an Fc gamma receptor selected from the group consisting ofFcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and/or FcγRIV. In a furtherembodiment, an anti-CD19 antibody of the invention is a bispecificantibody capable of specifically binding to human CD19 and FcγRIIB Inanother embodiment, an anti-CD19 antibody of the invention is abispecific antibody capable of specifically binding to human CD19 andhuman FcγRIIB

5.13. Variant Fc Regions

The present invention provides formulation of proteins comprising avariant Fc region. That is, a non naturally occurring Fc region, forexample an Fc region comprising one or more non naturally occurringamino acid residues. Also encompassed by the variant Fc regions ofpresent invention are Fc regions which comprise amino acid deletions,additions and/or modifications.

It will be understood that Fc region as used herein includes thepolypeptides comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain. Thus Fc refers to the lasttwo constant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat et al. (1991, NIH Publication91-3242, National Technical Information Service, Springfield, Va.). The“EU index as set forth in Kabat” refers to the residue numbering of thehuman IgG1 EU antibody as described in Kabat et al. supra. Fc may referto this region in isolation, or this region in the context of anantibody, antibody fragment, or Fc fusion protein. An Fc variant proteinmay be an antibody, Fc fusion, or any protein or protein domain thatcomprises an Fc region including, but not limited to, proteinscomprising variant Fc regions, which are non naturally occurringvariants of an Fc. Note: Polymorphisms have been observed at a number ofFc positions, including but not limited to Kabat 270, 272, 312, 315,356, and 358, and thus slight differences between the presented sequenceand sequences in the prior art may exist.

The present invention encompasses Fc variant proteins which have alteredbinding properties for an Fc ligand (e.g., an Fc receptor, Clq) relativeto a comparable molecule (e.g., a protein having the same amino acidsequence except having a wild type Fc region). Examples of bindingproperties include but are not limited to, binding specificity,equilibrium dissociation constant (K_(D)), dissociation and associationrates (k_(off) and k_(on) respectively), binding affinity and/oravidity. It is generally understood that a binding molecule (e.g., a Fcvariant protein such as an antibody) with a low K_(D) may be preferableto a binding molecule with a high K_(D). However, in some instances thevalue of the icon or koff may be more relevant than the value of theK_(D). One skilled in the art can determine which kinetic parameter ismost important for a given antibody application.

The affinities and binding properties of an Fc domain for its ligand maybe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art for determining Fc-FcγRinteractions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to, equilibrium methods (e.g., enzyme-linkedimmunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics(e.g., BIACORE® analysis), and other methods such as indirect bindingassays, competitive inhibition assays, fluorescence resonance energytransfer (FRET), gel electrophoresis and chromatography (e.g., gelfiltration). These and other methods may utilize a label on one or moreof the components being examined and/or employ a variety of detectionmethods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions.

In one embodiment, the Fc variant protein has enhanced binding to one ormore Fc ligand relative to a comparable molecule. In another embodiment,the Fc variant protein has an affinity for an Fc ligand that is at least2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold greater than that of a comparable molecule. In a specificembodiment, the Fc variant protein has enhanced binding to an Fcreceptor. In another specific embodiment, the Fc variant protein hasenhanced binding to the Fc receptor FcγRIIIA. In a further specificembodiment, the Fc variant protein has enhanced biding to the Fcreceptor FcγRIIB. In still another specific embodiment, the Fc variantprotein has enhanced binding to the Fc receptor FcRn. In yet anotherspecific embodiment, the Fc variant protein has enhanced binding to Clqrelative to a comparable molecule.

In one embodiment, an anti-CD19 antibody of the invention comprises avariant Fc domain wherein said variant Fc domain has enhanced bindingaffinity to Fc gamma receptor IIB relative to a comparable non-variantFc domain. In a further embodiment, an anti-CD19 antibody of theinvention comprises a variant Fc domain wherein said variant Fc domainhas an affinity for Fc gamma receptor IIB that is at least 2 fold, or atleast 3 fold, or at least 5 fold, or at least 7 fold, or a least 10fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, orat least 50 fold, or at least 60 fold, or at least 70 fold, or at least80 fold, or at least 90 fold, or at least 100 fold, or at least 200 foldgreater than that of a comparable non-variant Fc domain.

The serum half-life of proteins comprising Fc regions may be increasedby increasing the binding affinity of the Fc region for FcRn. In oneembodiment, the Fc variant protein has enhanced serum half life relativeto comparable molecule.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enables these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. Specific high-affinity IgGantibodies directed to the surface of target cells “arm” the cytotoxiccells and are absolutely required for such killing. Lysis of the targetcell is extracellular, requires direct cell-to-cell contact, and doesnot involve complement. It is contemplated that, in addition toantibodies, other proteins comprising Fc regions, specifically Fc fusionproteins, having the capacity to bind specifically to an antigen-bearingtarget cell will be able to effect cell-mediated cytotoxicity. Forsimplicity, the cell-mediated cytotoxicity resulting from the activityof an Fc fusion protein is also referred to herein as ADCC activity.

The ability of any particular Fc variant protein to mediate lysis of thetarget cell by ADCC can be assayed. To assess ADCC activity an Fcvariant protein of interest is added to target cells in combination withimmune effector cells, which may be activated by the antigen antibodycomplexes resulting in cytolysis of the target cell. Cytolysis isgenerally detected by the release of label (e.g. radioactive substrates,fluorescent dyes or natural intracellular proteins) from the lysedcells. Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Specificexamples of in vitro ADCC assays are described in Wisecarver et al.,1985 79:277-282; Bruggemann et al., 1987, J Exp Med 166:1351-1361;Wilkinson et al., 2001, J Immunol Methods 258:183-191; Patel et al.,1995 J Tmmunol Methods 184:29-38. ADCC activity of the Fc variantprotein of interest may also be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al., 1998, Proc. Natl. Acad.Sci. USA 95:652-656.

In one embodiment, an Fc variant protein has enhanced ADCC activityrelative to a comparable molecule. In a specific embodiment, an Fcvariant protein has ADCC activity that is at least 2 fold, or at least 3fold, or at least 5 fold or at least 10 fold or at least 50 fold or atleast 100 fold greater than that of a comparable molecule. In anotherspecific embodiment, an Fc variant protein has enhanced binding to theFc receptor FcγRIIIA and has enhanced ADCC activity relative to acomparable molecule. In other embodiments, the Fc variant protein hasboth enhanced ADCC activity and enhanced serum half life relative to acomparable molecule.

In one embodiment, an Fc variant protein has reduced ADCC activityrelative to a comparable molecule. In a specific embodiment, an Fcvariant protein has ADCC activity that is at least 2 fold, or at least 3fold, or at least 5 fold or at least 10 fold or at least 50 fold or atleast 100 fold lower than that of a comparable molecule. In anotherspecific embodiment, an Fc variant protein has reduced binding to the Fcreceptor FcγRIIIA and has reduced ADCC activity relative to a comparablemolecule. In other embodiments, the Fc variant protein has both reducedADCC activity and enhanced serum half life relative to a comparablemolecule.

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget cell in the presence of complement. The complement activationpathway is initiated by the binding of the first component of thecomplement system (Clq) to a molecule, an antibody for example,complexed with a cognate antigen. To assess complement activation, a CDCassay, e.g. as described in Gazzano-Santoro et al., 1996, J. lmmunol.Methods, 202:163, may be performed. In one embodiment, an Fc variantprotein has enhanced CDC activity relative to a comparable molecule. Ina specific embodiment, an Fc variant protein has CDC activity that is atleast 2 fold, or at least 3 fold, or at least 5 fold or at least 10 foldor at least 50 fold or at least 100 fold greater than that of acomparable molecule. In other embodiments, the Fc variant protein hasboth enhanced CDC activity and enhanced serum half life relative to acomparable molecule.

In one embodiment, the Fc variant protein has reduced binding to one ormore Fc ligand relative to a comparable molecule. In another embodiment,the Fc variant protein has an affinity for an Fc ligand that is at least2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold lower than that of a comparable molecule. In a specificembodiment, the Fc variant protein has reduced binding to an Fcreceptor. In another specific embodiment, the Fc variant protein hasreduced binding to the Fc receptor FcγRIIIA In a further specificembodiment, an Fc variant described herein has an affinity for the Fcreceptor FcγRIIIA that is at least about 5 fold lower than that of acomparable molecule, wherein said Fc variant has an affinity for the Fcreceptor FcγRIIB that is within about 2 fold of that of a comparablemolecule. In still another specific embodiment, the Fc variant proteinhas reduced binding to the Fc receptor FcRn. In yet another specificembodiment, the Fc variant protein has reduced binding to Clq relativeto a comparable molecule.

In one embodiment, the present invention provides Fc variants, whereinthe Fc region comprises a non naturally occurring amino acid residue atone or more positions selected from the group consisting of 234, 235,236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255,256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296,297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443as numbered by the EU index as set forth in Kabat. Optionally, the Fcregion may comprise a non naturally occurring amino acid residue atadditional and/or alternative positions known to one skilled in the art(see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT PatentPublications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO05/040217, WO 05/092925 and WO 06/020114).

In one embodiment, the present invention provides formulations, whereinthe Fc region comprises a non naturally occurring amino acid residue atone or more positions selected from the group consisting of 234, 235,236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255,256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296,297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443as numbered by the EU index as set forth in Kabat. Optionally, the Fcregion may comprise a non naturally occurring amino acid residue atadditional and/or alternative positions known to one skilled in the art(see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT PatentPublications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO05/040217, WO 05/092925 and WO 06/020114).

In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid residue selected from the group consisting of 234D, 234E,234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W,235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D,239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W,241 L, 241Y, 241E, 241 R. 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L,247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 2621, 262A, 262T, 262E,2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y,264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T, 2661,266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A,284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H,269G, 297S, 297D, 297E, 298H, 2981, 298T, 298F, 2991, 299L, 299A, 299S,299V, 299H, 299F, 299E, 3051, 313F, 316D, 325Q, 325L, 3251, 325D, 325E,325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E,328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K,330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 331G, 331A,331L, 331M, 331F, 331W, 331K, 331Q, 331E, 3315, 331V, 3311, 331C, 331Y,331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q,332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H,421K, 440Y and 434W as numbered by the EU index as set forth in Kabat.Optionally, the Fc region may comprise additional and/or alternative nonnaturally occurring amino acid residues known to one skilled in the art(see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT PatentPublications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752 and WO 05/040217).

In a specific embodiment, the present invention provides an Fc variantprotein formulation, wherein the Fc region comprises at least one nonnaturally occurring amino acid residue selected from the groupconsisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V,234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y,2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y,2401, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241 R. 243W, 243L 243Y,243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E,256M, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W,264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V,2651, 265L, 265H, 265T, 2661, 266A, 266T, 266M, 267Q, 267L, 268E, 269H,269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N,296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 2981, 298T,298F, 2991, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 3051, 313F, 316D,325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N,327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H,328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301,330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E,331S, 331V, 3311, 331C, 331Y, 331H, 331R, 331N, 331D, 3311, 332D, 332S,332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N,378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EUindex as set forth in Kabat. Optionally, the Fc region may compriseadditional and/or alternative non naturally occurring amino acidresidues known to one skilled in the art (see, e.g., U.S. Pat. Nos.5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

In another embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid at one or more positions selected from the group consistingof 239, 330 and 332, as numbered by the EU index as set forth in Kabat.In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 239D, 330L and 332E, asnumbered by the EU index as set forth in Kabat. Optionally, the Fcregion may further comprise additional non naturally occurring aminoacid at one or more positions selected from the group consisting of 252,254, and 256, as numbered by the EU index as set forth in Kabat. In aspecific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 239D, 330L and 332E, asnumbered by the EU index as set forth in Kabat and at least one nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 252Y, 254T and 256E, as numbered by the EU indexas set forth in Kabat.

In another embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid at one or more positions selected from the group consistingof 234, 235 and 331, as numbered by the EU index as set forth in Kabat.In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 234F, 235F, 235Y, and3315, as numbered by the EU index as set forth in Kabat. In a furtherspecific embodiment, an Fc variant of the invention comprises the 234F,235F, and 331S non naturally occurring amino acid residues, as numberedby the EU index as set forth in Kabat. In another specific embodiment,an Fc variant of the invention comprises the 234F, 235Y, and 3315 nonnaturally occurring amino acid residues, as numbered by the EU index asset forth in Kabat. Optionally, the Fc region may further compriseadditional non naturally occurring amino acid at one or more positionsselected from the group consisting of 252, 254, and 256, as numbered bythe EU index as set forth in Kabat. In a specific embodiment, thepresent invention provides an Fc variant, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 234F, 235F, 235Y, and 331S, as numbered by theEU index as set forth in Kabat; and at least one non naturally occurringamino acid at one or more positions are selected from the groupconsisting of 252Y, 254T and 256E, as numbered by the EU index as setforth in Kabat.

In another embodiment, the present invention provides an Fc variantprotein formulation, wherein the Fc region comprises at least a nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 239, 330 and 332, as numbered by the EU index asset forth in Kabat. In a specific embodiment, the present inventionprovides an Fc variant protein formulation, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 239D, 330L and 332E, as numbered by the EU indexas set forth in Kabat. Optionally, the Fc region may further compriseadditional non naturally occurring amino acid at one or more positionsselected from the group consisting of 252, 254, and 256, as numbered bythe EU index as set forth in Kabat. In a specific embodiment, thepresent invention provides an Fc variant protein formulation, whereinthe Fc region comprises at least one non naturally occurring amino acidselected from the group consisting of 239D, 330L and 332E, as numberedby the EU index as set forth in Kabat and at least one non naturallyoccurring amino acid at one or more positions are selected from thegroup consisting of 252Y, 254T and 256E, as numbered by the EU index asset forth in Kabat.

In another embodiment, the present invention provides an Fc variantprotein formulation, wherein the Fc region comprises at least one nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 234, 235 and 331, as numbered by the EU index asset forth in Kabat. In a specific embodiment, the present inventionprovides an Fc variant protein formulation, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 234F, 235F, 235Y, and 3315, as numbered by theEU index as set forth in Kabat. Optionally, the Fc region may furthercomprise additional non naturally occurring amino acid at one or morepositions selected from the group consisting of 252, 254, and 256, asnumbered by the EU index as set forth in Kabat. In a specificembodiment, the present invention provides an Fc variant proteinformulation, wherein the Fc region comprises at least one non naturallyoccurring amino acid selected from the group consisting of 234F, 235F,235Y, and 3315, as numbered by the EU index as set forth in Kabat; andat least one non naturally occurring amino acid at one or more positionsare selected from the group consisting of 252Y, 254T and 256E, asnumbered by the EU index as set forth in Kabat.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Ghetieet al., 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature332:563-564; Lund et al., 1991, J. Immunol 147:2657¬2662; Lund et al,1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation57:1537¬1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund etal., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al.,1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al.,2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. PatentPublication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO04/063351. Also encompassed by the present invention are Fc regionswhich comprise deletions, additions and/or modifications. Still othermodifications/substitutions/additions/deletions of the Fc domain will bereadily apparent to one skilled in the art.

Methods for generating non naturally occurring Fc regions are known inthe art. For example, amino acid substitutions and/or deletions can begenerated by mutagenesis methods, including, but not limited to,site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492(1985)), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guide to Methodsand Applications”, Academic Press, San Diego, pp. 177-183 (1990)), andcassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Preferably,site-directed mutagenesis is performed by the overlap-extension PCRmethod (Higuchi, in “PCR Technology: Principles and Applications for DNAAmplification”, Stockton Press, New York, pp. 61-70 (1989)). Thetechnique of overlap-extension PCR (Higuchi, ibid.) can also be used tointroduce any desired mutation(s) into a target sequence (the startingDNA). For example, the first round of PCR in the overlap-extensionmethod involves amplifying the target sequence with an outside primer(primer 1) and an internal mutagenesis primer (primer 3), and separatelywith a second outside primer (primer 4) and an internal primer (primer2), yielding two PCR segments (segments A and B). The internalmutagenesis primer (primer 3) is designed to contain mismatches to thetarget sequence specifying the desired mutation(s). In the second roundof PCR, the products of the first round of PCR (segments A and B) areamplified by PCR using the two outside primers (primers 1 and 4). Theresulting full-length PCR segment (segment C) is digested withrestriction enzymes and the resulting restriction fragment is clonedinto an appropriate vector. As the first step of mutagenesis, thestarting DNA (e.g., encoding an Fc fusion protein, an antibody or simplyan Fc region), is operably cloned into a mutagenesis vector. The primersare designed to reflect the desired amino acid substitution. Othermethods useful for the generation of variant Fc regions are known in theart (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. PatentPublication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO04/063351).

In some embodiments, an Fc variant protein comprises one or moreengineered glycoforms, i.e., a carbohydrate composition that iscovalently attached to the molecule comprising an Fc region. Engineeredglycoforms may be useful for a variety of purposes, including but notlimited to enhancing or reducing effector function. Engineeredglycoforms may be generated by any method known to one skilled in theart, for example by using engineered or variant expression strains, byco-expression with one or more enzymes, for example DIN-acetylglucosaminyltransferase III (GnTI11), by expressing a moleculecomprising an Fc region in various organisms or cell lines from variousorganisms, or by modifying carbohydrate(s) after the molecule comprisingFc region has been expressed. Methods for generating engineeredglycoforms are known in the art, and include but are not limited tothose described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davieset al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473)U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1;PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton,N.J.); GlycoMAb™ glycosylation engineering technology (GLYCARTbiotechnology AG, Zurich, Switzerland). See, e.g., WO 00061739;EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

It is contemplated that an Fc variant described herein may be generatedfrom, or a variant Fc region described herein may be introduced into anyantibody described in the art including but not limited toanti-fluorescein monoclonal antibody, 4-4-20 (Kranz et al., 1982 J.Biol. Chem. 257(12): 6987-6995), a humanized anti-TAG72 antibody (CC49)(Sha et al., 1994 Cancer Biother. 9(4): 341-9), an antibody thatspecifically bind an Eph Receptor including, but not limited to thosedisclosed in PCT Publication Nos. WO 04/014292, WO 03/094859 and U.S.patent application Ser. No. 10/863,729 (U.S. Pat. No. 7,604,799),antibodies that specifically bind Integrin αVβ3 including, but notlimited to, LM609 (Scripps), the murine monoclonal LM609 (PCTPublication WO 89/015155 and U.S. Pat. No. 5,753,230); the humanizedmonoclonal antibody MEDI-522 (a.k.a. VITAXIN®, MedImmune, Inc.,Gaithersburg, Md.; Wu et al., 1998, PNAS USA 95(11): 6037-6042; PCTPublications WO 90/33919 and WO 00/78815), an antibody againstinterferon alpha as disclosed in WO/2005/05059106, an antibody againstthe interferon receptor 1 as disclosed in WO/2006/059106, ERBITUX™(cetuximab) (also known as IMC-C225) (ImClone Systems Inc.), achimerized monoclonal antibody against EGFR; HERCEPTIN®. (Trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer; REOPRO®(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection. Other examples are ahumanized anti-CD18 F(ab′)2 (Genentech); CDP860 which is a humanizedanti-CD18 F(ab′)2 (Celltech, UK); PRO542 which is an anti-HIV gp120antibody fused with CD4 (Progenics/Genzyme Transgenics); C14 which is ananti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF IgG1 antibody(Genentech); OVAREX™ (Oregovomab) which is a murine anti-CA 125 antibody(Altarex); PANOREX™ (edrecolomab) which is a murine anti-17-IA cellsurface antigen IgG2a antibody (Glaxo Wellcome/Centocor); IMC-C225 whichis a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™ which isa humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); CAMPATH™ 1H/LDP-03 which is a humanized anti CD52IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN™ (rituximab) which is achimeric anti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE™ (epratuzumab) which is a humanized anti-CD22 IgG antibody(Immunomedics); Smart ID10 which is a humanized anti-HLA antibody(Protein Design Lab); ONCOLYM™ (Lym-1) is a radiolabelled murineanti-HLA DR antibody (Techniclone); anti-CD11a is a humanized IgG1antibody (Genentech/Xoma); ICM3 is a humanized anti-ICAM3 antibody (ICOSPharm); IDEC-114 is a primatized anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ (ipritumomab tiuxetan) is a radiolabelledmurine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanizedanti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (ProteinDesign Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody(Alexion Pharm); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDECPharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody(Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody(Celltech); LDP-02 is a humanized anti-α4β7 antibody(LeukoSite/Genentech); ORTHOCLONE™ OKT4A is a humanized anti-CD4 IgGantibody (Ortho Biotech); ANTOVA™ (ruplizumab) is a humanized anti-CD40LIgG antibody (Biogen); ANTEGREN™ (natalizumab) is a humanized anti-VLA-4IgG antibody (Elan); MDX-33 is a human anti-CD64 (FcγR) antibody(Medarex/Centeon); rhuMab-E25 is a humanized anti-IgE IgG1 antibody(Genentech/Norvartis/Tanox Biosystems); IDEC-152 is a primatizedanti-CD23 antibody (IDEC Pharm); ABX-CBL is a murine anti CD-147 IgMantibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody(Medimmune/Bio Transplant); ORTHOCLONE-OKT3™ (muromonab-CD3) is a murineanti-CD3 IgG2a antibody (ortho Biotech); SIMULECT™ (basiliximab) is achimeric anti-CD25 IgG1 antibody (Novartis Pharm); LDP-01 is a humanizedanti-β2-integrin IgG antibody (LeukoSite); Anti-LFA-1 is a murine antiCD18 F(ab′)2 (Pasteur-Mericex/Immunotech); CAT-152 is a humananti-TGF-β2 antibody (Cambridge Ab Tech); and Corsevin M is a chimericanti-Factor VII antibody (Centocor).

Additional antibodies which may comprise an Fc variant region describedherein may specifically bind a cancer or tumor antigen for example,including, but not limited to, KS 1/4 pan-carcinoma antigen (Perez andWalker, 1990, J. Immunol. 142: 3662-3667; Bumal, 1988, Hybridoma 7(4):407-415), ovarian carcinoma antigen (CA125) (Yu et al., 1991, CancerRes. 51(2): 468-475), prostatic acid phosphate (Tailor et al., 1990,Nucl. Acids Res. 18(16): 4928), prostate specific antigen (Henttu andVihko, 1989, Biochem. Biophys. Res. Comm. 160(2): 903-910; Israeli etal., 1993, Cancer Res. 53: 227-230), melanoma-associated antigen p97(Estin et al., 1989, J. Natl. Cancer Instit. 81(6): 445-446), melanomaantigen gp75 (Vijayasardahl et al., 1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali etal., 1987, Cancer 59: 55-63; Mittelman et al., 1990, J. Clin. Invest.86: 2136-2144), prostate specific membrane antigen, carcinoembryonicantigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol. 13: 294),polymorphic epithelial mucin antigen, human milk fat globule antigen,colorectal tumor-associated antigens such as: CEA, TAG-72 (Yokata etal., 1992, Cancer Res. 52: 3402-3408), C017-1A (Ragnhammar et al., 1993,Tnt. J. Cancer 53: 751-758); GICA 19-9 (Herlyn et al., 1982, J. Clin.Immunol. 2: 135), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19(Ghetie et al., 1994, Blood 83: 1329-1336), human B-lymphomaantigen-CD20 (Reff et al., 1994, Blood 83:435-445), CD33 (Sgouros etal., 1993, J. Nucl. Med. 34:422-430), melanoma specific antigens such asganglioside GD2 (Saleh et al., 1993, J. Immunol., 151, 3390-3398),ganglioside GD3 (Shitara et al., 1993, Cancer Immunol. Immunother.36:373-380), ganglioside GM2 (Livingston et al., 1994, J. Clin. Oncol.12: 1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:5244-5250), tumor-specific transplantation type of cell-surface antigen(TSTA) such as virally-induced tumor antigens including T-antigen DNAtumor viruses and Envelope antigens of RNA tumor viruses, oncofetalantigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetalantigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46: 3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185HER2), polymorphicepithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci.17:359), malignant human lymphocyte antigen-APO-1 (Bernhard et al.,1989, Science 245: 301-304), differentiation antigen (Feizi, 1985,Nature 314: 53-57) such as T antigen found in fetal erythrocytes,primary endoderm I antigen found in adult erythrocytes, preimplantationembryos, I(Ma) found in gastric adenocarcinomas, M18, M39 found inbreast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl,VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H),C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma,AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinomacells, TL5 (blood group A), EGF receptor found in A431 cells, E1 series(blood group B) found in pancreatic cancer, FC10.2 found in embryonalcarcinoma cells, gastric adenocarcinoma antigen, CO-514 (blood groupLea) found in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43(blood group Leb), G49 found in EGF receptor of A431 cells, MH2 (bloodgroup ALeb/Ley) found in colonic adenocarcinoma, 19.9 found in coloncancer, gastric cancer mucins, T5A7 found in myeloid cells, R24 found inmelanoma, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8 foundin embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in 4 to 8-cellstage embryos. In one embodiment, the antigen is a T cell receptorderived peptide from a Cutaneous Tcell Lymphoma (see, Edelson, 1998, TheCancer Journal 4:62).

An Fc variant described herein may be generated from, or a variant Fcregion described herein may be introduced into any antibody.Furthermore, a variant Fc region described herein may be utilized togenerate an Fc fusion protein. Accordingly, virtually any molecule maybe targeted by and/or incorporated into an antibody and/or Fc fusionprotein comprising an Fc variant described herein including, but notlimited to, the following list of proteins, as well as subunits,domains, motifs and epitopes belonging to the following list ofproteins: renin; a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor VII, factor VIIIC, factor IX, tissue factor (TF), and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a scrum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors such as,for example, EGFR, VEGFR; interferons such as alpha interferon (a-IFN),beta interferon 0(3-IFN) and gamma interferon (y-IFN); interferonreceptor components such as interferon receptor 1; protein A or D;rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor; platelet-derived growth factor(PDGF); fibroblast growth factor such as αFGF and βFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-alpha andTGF-beta, including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-5; insulin-likegrowth factor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I (brainIGF-I), insulin-like growth factor binding proteins; CD proteins such asCD2, CD3, CD4, CD 8, CD11 a, CD14, CD18, CD19, CD20, CD22, CD23, CD25,CD33, CD34, CD40, CD40L, CD52, CD63, CD64, CD80 and CD147;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such asinterferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs),such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 toIL-13; TNFa, HMGB1; HMGB2; superoxide dismutase; T-cell receptors;surface membrane proteins; decay accelerating factor; viral antigen suchas, for example, a portion of the AIDS envelope, e.g., gp120; transportproteins; homing receptors; addressins; regulatory proteins; celladhesion molecules such as LFA-1, Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3and VCAM, a4/p7 integrin, and (Xv/p3 integrin including either a orsubunits thereof, integrin alpha subunits such as CD49a, CD49b, CD49c,CD49d, CD49e, CD49f, alpha7, alpha8, alpha9, alphaD, CD11 a, CD11b,CD51, CD11 c, CD41, alphallb, alphalELb; integrin beta subunits such as,CD29, CD 18, CD61, CD104, beta5, beta6, beta7 and beta8; Integrinsubunit combinations including but not limited to, αVβ3, αVβ5 and α4β7;a member of an apoptosis pathway; IgE; blood group antigens; flk2/flt3receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C; achitinase or chitinase-like molecule such as YKL-40 and AMCase; an Ephreceptor such as EphA2, EphA4, EphB2, etc.; a Human Leukocyte Antigen(HLA) such as HLA-DR; complement proteins such as complement receptorCR1, ClRq and other complement factors such as C3, and C5; aglycoprotein receptor such as Gplbα, GPIIb/IIIa and CD200;co-stimulatory molecules such as CD28/CTLA-4, ICOS/AILIM, PD-1.

Additional molecules which may comprise a variant Fc region describedherein are those that specifically bind cancer antigens including, butnot limited to, ALK receptor (pleiotrophin receptor), pleiotrophin, KS1/4 pan-carcinoma antigen; ovarian carcinoma antigen (CA125); prostaticacid phosphate; prostate specific antigen (PSA); melanoma-associatedantigen p97; melanoma antigen gp75; high molecular weight melanomaantigen (HMW-MAA); prostate specific membrane antigen; carcinoembryonicantigen (CEA); polymorphic epithelial mucin antigen; human milk fatglobule antigen; colorectal tumor-associated antigens such as: CEA,TAG-72, C017-1A, GICA 19-9, CTA-1 and LEA; Burkitt's lymphomaantigen-38.13; CD19; human B-lymphoma antigen-CD20; CD33; melanomaspecific antigens such as ganglioside GD2, ganglioside GD3, gangliosideGM2 and ganglioside GM3; tumor-specific transplantation typecell-surface antigen (TSTA); virally-induced tumor antigens includingT-antigen, DNA tumor viruses and Envelope antigens of RNA tumor viruses;oncofetal antigen-alpha-fetoprotein such as CEA of colon, 5T4 oncofetaltrophoblast glycoprotein and bladder tumor oncofetal antigen;differentiation antigen such as human lung carcinoma antigens L6 andL20; antigens of fibrosarcoma; human leukemia T cell antigen-Gp37;neoglycoprotein; sphingolipids; breast cancer antigens such as EGFR(Epidermal growth factor receptor); NY-BR-16; NY-BR-16 and HER2 antigen(p185HER2); polymorphic epithelial mucin (PEM); malignant humanlymphocyte antigen-APO-1; differentiation antigen such as 1 antigenfound in fetal erythrocytes; primary endoderm I antigen found in adulterythrocytes; preimplantation embryos; I(Ma) found in gastricadenocarcinomas; M18, M39 found in breast epithelium; SSEA-1 found inmyeloid cells; VEP8; VEP9; Myl; VIM-D5; D156-22 found in colorectalcancer; TRA-1-85 (blood group H); SCP-1 found in testis and ovariancancer; C14 found in colonic adenocarcinoma; F3 found in lungadenocarcinoma; AH6 found in gastric cancer; Y hapten; Ley found inembryonal carcinoma cells; TL5 (blood group A); EGF receptor found inA431 cells; E1 series (blood group B) found in pancreatic cancer; FC10.2found in embryonal carcinoma cells; gastric adenocarcinoma antigen;CO-514 (blood group Lea) found in Adenocarcinoma; NS-10 found inadenocarcinomas; CO-43 (blood group Leb); G49 found in EGF receptor ofA431 cells; MH2 (blood group ALeb/Ley) found in colonic adenocarcinoma;19.9 found in colon cancer; gastric cancer mucins; T5A7 found in myeloidcells; R24 found in melanoma; 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2,and M1:22:25:8 found in embryonal carcinoma cells and SSEA-3 and SSEA-4found in 4 to 8-cell stage embryos; Cutaneous Tcell Lymphoma antigen;MART-1 antigen; Sialy Tn (STn) antigen; Colon cancer antigen NY-CO-45;Lung cancer antigen N Y-LU-12 variant A; Adenocarcinoma antigen ART1;Paraneoplastic associated brain-testis-cancer antigen (onconeuronalantigen MA2; paraneoplastic neuronal antigen); Neuro-oncological ventralantigen 2 (NOVA2); Hepatocellular carcinoma antigen gene 520;TUMOR-ASSOCIATED ANTIGEN CO-029; Tumor-associated antigens MAGE-C1(cancer/testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6),MAGE-2, MAGE-4a, MAGE-4b and MAGE-X2; Cancer-Testis Antigen (NY-EOS-1);YKL-40 and fragments of any of the above-listed polypeptides.

5.14. Glycosylation of Antibodies

In still another embodiment, the glycosylation of antibodies utilized inaccordance with the invention is modified. For example, an aglycoslatedantibody can be made (i.e., the antibody lacks glycosylation).Glycosylation can be altered to, for example, increase the affinity ofthe antibody for a target antigen. Such carbohydrate modifications canbe accomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence. For example, one or moreamino acid substitutions can be made that result in elimination of oneor more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. Such an approach is describedin further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861. One or moreamino acid substitutions can also be made that result in elimination ofa glycosylation site present in the Fc region (e.g., Asparagine 297 ofIgG). Furthermore, aglycosylated antibodies may be produced in bacterialcells which lack the necessary glycosylation machinery.

An antibody can also be made that has an altered type of glycosylation,such as a hypofucosylated antibody having reduced amounts of fucosylresidues or an antibody having increased bisecting GlcNAc structures.Such altered glycosylation patterns have been demonstrated to increasethe ADCC ability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, U.S. Pat. No. 6,946,292;European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO99/54342 each of which is incorporated herein by reference in itsentirety.

5.15. Engineering Effector Function

It may be desirable to modify an anti-CD19 antibody of the inventionwith respect to effector function, so as to enhance the effectiveness ofthe antibody in treating B cell malignancies, for example. For example,cysteine residue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and/orantibody-dependent cellular cytotoxicity (ADCC). See, Caron et al., J.Exp Med., 176:1191-1195 (1992) and Shopes, B., J. Immunol.,148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research, 53:2560-2565 (1993). Anantibody can also be engineered which has dual Fc regions and maythereby have enhanced complement lysis and ADCC capabilities. See,Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989).

Other methods of engineering Fc regions of antibodies so as to altereffector functions are known in the art (e.g., U.S. Patent PublicationNo. 20040185045 and PCT Publication No. WO 2004/016750, both to Koeniget al., which describe altering the Fc region to enhance the bindingaffinity for FcγRIIB as compared with the binding affinity for FcγRIIA;see, also, PCT Publication Nos. WO 99/58572 to Armour et al., WO99/51642 to ldusogie et al., and U.S. Pat. No. 6,395,272 to Deo et al.;the disclosures of which are incorporated herein in their entireties).Methods of modifying the Fc region to decrease binding affinity toFcγRIIB are also known in the art (e.g., U.S. Patent Publication No.20010036459 and PCT Publication No. WO 01/79299, both to Ravetch et al.,the disclosures of which are incorporated herein in their entireties).Modified antibodies having variant Fc regions with enhanced bindingaffinity for FcγRIIIA and/or FcγRIIA as compared with a wildtype Fcregion have also been described (e.g., PCT Publication Nos. WO2004/063351, to Stavenhagen et al., the disclosure of which isincorporated herein in its entirety).

In vitro assays known in the art can be used to determine whetheranti-CD19 antibodies used in compositions and methods of the inventionare capable of mediating ADCC, such as those described herein.

5.16. Manufacture/Production of Anti-CD19 Antibodies

Once a desired anti-CD19 antibody is engineered, the anti-CD19 antibodycan be produced on a commercial scale using methods that are well-knownin the art for large scale manufacturing of antibodies. For example,this can be accomplished using recombinant expressing systems such as,but not limited to, those described below.

5.17. Recombinant Expression Systems

Recombinant expression of an antibody or variant thereof, generallyrequires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof, has been obtained, the vector for the production of theantibody molecule may be produced by recombinant DNA technology usingtechniques well-known in the art. See, e.g., U U.S. Pat. No. 6,331,415,which is incorporated herein by reference in its entirety. Thus, methodsfor preparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well-known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule, a heavy or light chain of an antibody, aheavy or light chain variable domain of an antibody or a portionthereof, or a heavy or light chain CDR, operably linked to a promoter.Such vectors may include the nucleotide sequence encoding the constantregion of the antibody molecule (see, e.g., International PublicationNos. WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464) and thevariable domain of the antibody may be cloned into such a vector forexpression of the entire heavy, the entire light chain, or both theentire heavy and light chains.

In another embodiment, anti-CD19 antibodies can be made using targetedhomologous recombination to produce all or portions of the anti-CD19antibodies (see, U.S. Pat. Nos. 6,063,630, 6,187,305, and 6,692,737). Incertain embodiments, anti-CD19 antibodies can be made using randomrecombination techniques to produce all or portions of the anti-CD19antibodies (see, U.S. Pat. Nos. 6,361,972, 6,524,818, 6,541,221, and6,623,958). Anti-CD19 antibodies can also be produced in cellsexpressing an antibody from a genomic sequence of the cell comprising amodified immunoglobulin locus using Cre-mediated site-specifichomologous recombination (see, U.S. Pat. No. 6,091,001). The host cellline may be derived from human or nonhuman species including but notlimited to mouse, and Chinese hamster. Where human or humanized antibodyproduction is desired, the host cell line should be a human cell line.These methods may advantageously be used to engineer stable cell lineswhich permanently express the antibody molecule.

Once the expression vector is transferred to a host cell by conventionaltechniques, the transfected cells are then cultured by conventionaltechniques to produce an antibody. Thus, the invention includes hostcells containing a polynucleotide encoding an antibody of the inventionor fragments thereof, or a heavy or light chain thereof, or portionthereof, or a single-chain antibody of the invention, operably linked toa heterologous promoter. In certain embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressan anti-CD19 antibody or portions thereof that can be used in theengineering and generation of anti-CD19 antibodies (see, e.g., U.S. Pat.No. 5,807,715). For example, mammalian cells such as Chinese hamsterovary cells (CHO), in conjunction with a vector such as the majorintermediate early gene promoter element from human cytomegalovirus isan effective expression system for antibodies (Foecking et al., Gene,45:101 (1986); and Cockett et al., Bio/Technology, 8:2 (1990)). Inaddition, a host cell strain may be chosen which modulates theexpression of inserted antibody sequences, or modifies and processes theantibody gene product in the specific fashion desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the antibody or portionthereof expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO(a murine myeloma cell line that does not endogenously produce anyfunctional immunoglobulin chains), CRL7030 and HsS78Bst cells.

In one embodiment, human cell lines developed by immortalizing humanlymphocytes can be used to recombinantly produce monoclonal humananti-CD19 antibodies. In one embodiment, the human cell line PER.C6.(Crucell, Netherlands) can be used to recombinantly produce monoclonalhuman anti-CD19 antibodies.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions comprising an anti-CD19 antibody, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 1989, J.Biol. Chem., 24:5503-5509 (1989)); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione-S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to glutathione-agarose affinity matrix followed by elution inthe presence of free glutathione. The pGEX vectors are designed tointroduce athrombin and/or factor Xa protease cleavage sites into theexpressed polypeptide so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpocloptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example, the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample, the polyhedrin promoter).

In mammalian host cells, a number of virus based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion into a non-essential region of the viral genome (e.g., regionE1 or E3) will result in a recombinant virus that is viable and capableof expressing the antibody molecule in infected hosts (e.g., see, Logan& Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon should generally be in frame with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see, e.g., Bittneret al., Methods in Enzymol., 153:51-544(1987)).

Stable expression can be used for long-term, high-yield production ofrecombinant proteins. For example, cell lines which stably express theantibody molecule may be generated. Host cells can be transformed withan appropriately engineered vector comprising expression controlelements (e.g., promoter, enhancer, transcription terminators,polyadenylation sites, etc.), and a selectable marker gene. Followingthe introduction of the foreign DNA, cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells that stably integrated theplasmid into their chromosomes to grow and form foci which in turn canbe cloned and expanded into cell lines. Plasmids that encode ananti-CD19 antibody can be used to introduce the gene/cDNA into any cellline suitable for production in culture.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell,11:223 (1977)), hypoxanthineguanine phosphoribosyltransferase (Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell, 22:8-17 (1980)) genes canbe employed in tk⁻, hgprt⁻ or aprrcells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); andMorgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH11(5):155-2 15 (1993)); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, N Y (1993); Kricgler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in HumanGenetics, John Wiley & Sons, N Y (1994); Colberre-Garapin et al., 1981,J. Mol. Biol., 150:1, which are incorporated by reference herein intheir entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see, Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. Academic Press, New York(1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol., 3:257(1983)). Antibody expression levels may be amplified through the userecombinant methods and tools known to those skilled in the art ofrecombinant protein production, including technologies that remodelsurrounding chromatin and enhance transgene expression in the form of anactive artificial transcriptional domain.

The host cell may be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors maycontain identical or different selectable markers. A single vector whichencodes, and is capable of expressing, both heavy and light chainpolypeptides may also be used. In such situations, the light chainshould be placed 5′ to the heavy chain to avoid an excess of toxic freeheavy chain (Proudfoot, Nature 322:562-65 (1986); and Kohler, 1980,Proc. Natl. Acad. Sci. USA, 77:2197 (1980)). The coding sequences forthe heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule has been produced by recombinant expression,it may be purified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigensProtein A or Protein G, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. Further, the antibodies ofthe present invention or fragments thereof may be fused to heterologouspolypeptide sequences described herein or otherwise known in the art tofacilitate purification.

5.17.1. Antibody Purification and Isolation

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology, 10:163-167 (1992) describe a procedure forisolating antibodies which are secreted into the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibodymutant is secreted into the medium, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, for example, an Amicon or MilliporePcllicon ultrafiltration unit. A protease inhibitor such as PMSF may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, ion exchange chromatography, gel electrophoresis,dialysis, and/or affinity chromatography either alone or in combinationwith other purification steps. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody mutant. Protein A can be usedto purify antibodies that are based on human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Methods, 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBO1, 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin, SEPHAROSE chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, and performed at low salt concentrations (e.g.,from about 0-0.25 M salt).

5.18. Therapeutic Anti-CD19 Antibodies

An anti-CD19 antibody used in compositions and methods of the inventionmay be a human antibody or a humanized antibody that may mediate B celllineage apoptosis and/or human ADCC, or can be selected from knownanti-CD19 antibodies that may mediate B lineage cell apoptosis and/orhuman ADCC. In certain embodiments, anti-CD19 antibodies can be chimericantibodies. In certain embodiments, an anti-CD19 antibody can be amonoclonal human, humanized, or chimeric anti-CD19 antibody. Ananti-CD19 antibody used in compositions and methods of the invention maybe a human antibody or a humanized antibody of the IgG1 or IgG3 humanisotype or any IgG1 or IgG3 allele found in the human population. Inother embodiments, an anti-CD19 antibody used in compositions andmethods of the invention can be a human antibody or a humanized antibodyof the IgG2 or IgG4 human isotype or any IgG2 or IgG4 allele found inthe human population.

While such antibodies can be generated using the techniques describedabove, in other embodiments of the invention, the murine antibodiesHB12A and HB12B as described herein or other commercially availableanti-CD19 antibodies can be chimerized, humanized, or made into humanantibodies.

For example, known anti-CD19 antibodies that can be used include, butare not limited to, HD37 (IgG1, kappa) (DAKO North America, Inc,Carpinteria, Calif.), BU12 (Callard et al., J. Immunology,148(10):2983-7 (1992)), 4G7 (IgG1) (Meeker et al., Hybridoma,3(4):305-20 (1984 Winter)), J4.119 (Beckman Coulter, Krefeld, Germany),B43 (PharMingen, San Diego, Calif.), SJ25C1 (BD PharMingen, San Diego,Calif.), FMC63 (IgG2a) (Zola et al., Immunol. Cell. Biol. 69(PT6):411-22 (1991); Nicholson et al., Mol. Immunol., 34:1157-1165 (1997);Pietersz et al., Cancer Immunol. Immunotherapy, 41:53-60 (1995)),89B(B4) (IgG1) (Beckman Coulter, Miami, Fla.; Nadler et al., J.Immunol., 131:244-250 (1983)), and/or HD237 (IgG2b) (FourthInternational Workshop on Human Leukocyte Differentiation Antigens,Vienna, Austria, 1989; and Pezzutto et al., J. Immunol.,138(9):2793-2799 (1987)).

In certain embodiments, the antibody is an isotype switched variant of aknown antibody (e.g., to an IgG1 or IgG3 human isotype) such as thosedescribed above.

An anti-CD19 antibodies used in compositions and methods of theinvention can be naked antibodies, immunoconjugates or fusion proteins.Anti-CD19 antibodies described above for use in compositions and methodsof the invention may be able to reduce or deplete B cells andcirculating immunoglobulin in a human treated therewith. Depletion of Bcells can be in circulating B cells, or in particular tissues such as,but not limited to, bone marrow, spleen, gut-associated lymphoidtissues, and/or lymph nodes. Such depletion may be achieved via variousmechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC),and/or by blocking of CD19 interaction with its intended ligand, and/orcomplement dependent cytotoxicity (CDC), inhibition of B cellproliferation and/or induction of B cell death (e.g., via apoptosis). By“depletion” of B cells it is meant a reduction in circulating B cellsand/or B cells in particular tissue(s) by at least about 25%, 40%, 50%,65%, 75%, 80%, 85%, 90%, 95% or more. In particular embodiments,virtually all detectable B cells are depleted from the circulationand/or particular tissue(s). By “depletion” of circulatingimmunoglobulin (Ig) it is meant a reduction by at least about 25%, 40%,50%, 65%, 75%, 80%, 85%, 90%, 95% or more. In particular embodiments,virtually all detectable Ig is depleted from the circulation.

5.18.1. Screening of Antibodies for Human CD19 Binding

Binding assays can be used to identify antibodies that bind the humanCD19 antigen. Binding assays may be performed either as direct bindingassays or as competition-binding assays. Binding can be detected usingstandard ELISA or standard Flow Cytometry assays. In a direct bindingassay, a candidate antibody is tested for binding to human CD19 antigen.In certain embodiments, the screening assays comprise, in a second step,determining the ability to cause cell death or apoptosis of B cellsexpressing human CD19. Competition-binding assays, on the other hand,assess the ability of a candidate antibody to compete with a knownanti-CD19 antibody or other compound that binds human CD19.

In a direct binding assay, the human CD19 antigen is contacted with acandidate antibody under conditions that allow binding of the candidateantibody to the human CD19 antigen. The binding may take place insolution or on a solid surface. The candidate antibody may have beenpreviously labeled for detection. Any detectable compound can be usedfor labeling, such as, but not limited to, a luminescent, fluorescent,or radioactive isotope or group containing same, or a nonisotopic label,such as an enzyme or dye. After a period of incubation sufficient forbinding to take place, the reaction is exposed to conditions andmanipulations that remove excess or non-specifically bound antibody.Typically, it involves washing with an appropriate buffer. Finally, thepresence of a CD19-antibody complex is detected.

In a competition-binding assay, a candidate antibody is evaluated forits ability to inhibit or displace the binding of a known anti-CD19antibody (or other compound) to the human CD19 antigen. A labeled knownbinder of CD19 may be mixed with the candidate antibody, and placedunder conditions in which the interaction between them would normallyoccur, with and without the addition of the candidate antibody. Theamount of labeled known binder of CD19 that binds the human CD19 may becompared to the amount bound in the presence or absence of the candidateantibody.

In one embodiment, the binding assay is carried out with one or morecomponents immobilized on a solid surface to facilitate antibody antigencomplex formation and detection. In various embodiments, the solidsupport could be, but is not restricted to, polyvinylidene fluoride,polycarbonate, polystyrene, polypropylene, polyethylene, glass,nitrocellulose, dextran, nylon, polyacrylamide and agarose. The supportconfiguration can include beads, membranes, microparticles, the interiorsurface of a reaction vessel such as a microtiter plate, test tube orother reaction vessel. The immobilization of human CD19, or othercomponent, can be achieved through covalent or non-covalent attachments.In one embodiment, the attachment may be indirect, i.e., through anattached antibody. In another embodiment, the human CD19 antigen andnegative controls are tagged with an epitope, such as glutathioneS-transferase (GST) so that the attachment to the solid surface can bemediated by a commercially available antibody such as anti-GST (SantaCruz Biotechnology).

For example, such an affinity binding assay may be performed using thehuman CD19 antigen which is immobilized to a solid support. Typically,the non-mobilized component of the binding reaction, in this case thecandidate anti-CD19 antibody, is labeled to enable detection. A varietyof labeling methods are available and may be used, such as luminescent,chromophore, fluorescent, or radioactive isotope or group containingsame, and nonisotopic labels, such as enzymes or dyes. In oneembodiment, the candidate anti-CD19 antibody is labeled with afluorophore such as fluorescein isothiocyanate (FITC, available fromSigma Chemicals, St. Louis). Such an affinity binding assay may beperformed using the human CD19 antigen immobilized on a solid surface.Anti-CD19 antibodies are then incubated with the antigen and thespecific binding of antibodies is detected by methods known in the artincluding, but not limited to, SiaCore Analyses, ELISA, FMET and R1Amethods.

Finally, the label remaining on the solid surface may be detected by anydetection method known in the art. For example, if the candidateanti-CD19 antibody is labeled with a fluorophore, a fluorimeter may beused to detect complexes.

The human CD19 antigen can be added to binding assays in the form ofintact cells that express human CD19 antigen, or isolated membranescontaining human CD19 antigen. Thus, direct binding to human CD19antigen may be assayed in intact cells in culture or in animal models inthe presence and absence of the candidate anti-CD19 antibody. A labeledcandidate anti-CD19 antibody may be mixed with cells that express humanCD19 antigen, or with crude extracts obtained from such cells, and thecandidate anti-CD19 antibody may be added. Isolated membranes may beused to identify candidate anti-CD19 antibodies that interact with humanCD19. For example, in a typical experiment using isolated membranes,cells may be genetically engineered to express human CD19 antigen.Membranes can be harvested by standard techniques and used in an invitro binding assay. Labeled candidate anti-CD19 antibody (e.g.,fluorescent labeled antibody) is bound to the membranes and assayed forspecific activity; specific binding is determined by comparison withbinding assays performed in the presence of excess unlabeled (cold)candidate anti-CD19 antibody. Soluble human CD19 antigen may also berecombinantly expressed and utilized in non-cell based assays toidentify antibodies that bind to human CD19 antigen. The recombinantlyexpressed human CD19 polypeptides can be used in the non-cell basedscreening assays. Peptides corresponding to one or more of the bindingportions of human CD19 antigen, or fusion proteins containing one ormore of the binding portions of human CD19 antigen can also be used innon-cell based assay systems to identify antibodies that bind toportions of human CD19 antigen. In non-cell based assays therecombinantly expressed human CD19 is attached to a solid substrate suchas a test tube, microliter well or a column, by means well-known tothose in the art (see, Ausubel et al., supra). The test antibodies arethen assayed for their ability to bind to human CD19 antigen.

The binding reaction may also be carried out in solution. In this assay,the labeled component is allowed to interact with its binding partner(s)in solution. If the size differences between the labeled component andits binding partner(s) permit such a separation, the separation can beachieved by passing the products of the binding reaction through anultrafilter whose pores allow passage of unbound labeled component butnot of its binding partner(s) or of labeled component bound to itspartner(s). Separation can also be achieved using any reagent capable ofcapturing a binding partner of the labeled component from solution, suchas an antibody against the binding partner and so on.

In one embodiment, for example, a phage library can be screened bypassing phage from a continuous phage display library through a columncontaining purified human CD19 antigen, or derivative, analog, fragment,or domain, thereof, linked to a solid phase, such as plastic beads. Byaltering the stringency of the washing buffer, it is possible to enrichfor phage that express peptides with high affinity for human CD19antigen. Phage isolated from the column can be cloned and affinities canbe measured directly. Knowing which antibodies and their amino acidsequences confer the strongest binding to human CD19 antigen, computermodels can be used to identify the molecular contacts between CD19antigen and the candidate antibody.

In another specific embodiment, the solid support is membrane containinghuman CD19 antigen attached to a microtiter dish. Candidate antibodies,for example, can bind cells that express library antibodies cultivatedunder conditions that allow expression of the library members in themicroliter dish. Library members that bind to the human CD19 areharvested. Such methods, are generally described by way of example inParmley and Smith, 1988, Gene, 73:305-318; Fowlkes et al., 1992,BioTechniques, 13:422-427; PCT Publication No. WO94/18318; and inreferences cited hereinabove. Antibodies identified as binding to humanCD19 antigen can be of any of the types or modifications of antibodiesdescribed above.

5.18.2. Screening of Antibodies for Human ADCC Effector Function

Antibodies of the human IgG class, which have functional characteristicssuch a long half-life in serum and the ability to mediate variouseffector functions are used in certain embodiments of the invention(Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc.,Chapter 1 (1995)). The human IgG class antibody is further classifiedinto the following 4 subclasses: IgG1, IgG2, IgG3 and IgG4. A largenumber of studies have so far been conducted for ADCC and CDC aseffector functions of the IgG class antibody, and it has been reportedthat among antibodies of the human IgG class, the IgG1 subclass has thehighest ADCC activity and CDC activity in humans (Chemical Immunology,65, 88 (1997)).

Expression of ADCC activity and CDC activity of the human lgG1 subclassantibodies generally involves binding of the Fc region of the antibodyto a receptor for an antibody (hereinafter referred to as “FcγR”)existing on the surface of effector cells such as killer cells, naturalkiller cells or activated macrophages. Various complement components canbe bound. Regarding the binding, it has been suggested that severalamino acid residues in the hinge region and the second domain of Cregion (hereinafter referred to as “Cγ2 domain”) of the antibody areimportant (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319(1995), Chemical Immunology, 65, 88 (1997)) and that a sugar chain inthe Cγ2 domain (Chemical Immunology, 65, 88 (1997)) is also important.

Anti-CD19 antibodies can be modified with respect to effector function,e.g., so as to enhance ADCC and/or complement dependent cytotoxicity(CDC) of the antibody. This may be achieved by introducing one or moreamino acid substitutions in the Fc region of an antibody. Cysteineresidue(s) may also be introduced in the Fc region, allowing forinterchain disulfide bond formation in this region. In this way ahomodimeric antibody can be generated that may have improvedinternalization capability and or increased complement-mediated cellkilling and ADCC (Caron et al., J. Exp. Med., 176:1191-1195 (1992) andShopes, J. Immunol., 148:2918-2922 (1992)). Heterobifunctionalcross-linkers can also be used to generate homodimeric antibodies withenhanced anti-tumor activity (Wolff et al., Cancer Research,53:2560-2565 (1993)). Antibodies can also be engineered to have two ormore Fc regions resulting in enhanced complement lysis and ADCCcapabilities (Stevenson et al., Anti-Cancer Drug Design, (3)219-230(1989)).

Other methods of engineering Fc regions of antibodies so as to altereffector functions are known in the art (e.g., U.S. Patent PublicationNo. 20040185045 and PCT Publication No. WO 2004/016750, both to Koeniget al., which describe altering the Fc region to enhance the bindingaffinity for FcγRIIB as compared with the binding affinity for FCγRIIA;see also PCT Publication Nos. WO 99/58572 to Armour et al., WO 99/51642to Idusogic et al., and U.S. Pat. No. 6,395,272 to Dco et al.; thedisclosures of which are incorporated herein in their entireties).Methods of modifying the Fc region to decrease binding affinity toFcγRIIB are also known in the art (e.g., U.S. Patent Publication No.20010036459 and PCT Publication No. WO 01/79299, both to Ravetch et al.,the disclosures of which are incorporated herein in their entireties).Modified antibodies having variant Fc regions with enhanced bindingaffinity for FcγRIIIA and/or FcγRIIA as compared with a wildtype Fcregion have also been described (e.g., PCT Publication No. WO2004/063351, to Stavenhagen et al.; the disclosure of which isincorporated herein in its entirety).

At least four different types of FcγR have been found, which arerespectively called FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), andFcγRIV. In human, FcγRII and FcγRIII are further classified into FcγRIIaand FcγRIIb, and FcγRIIIa and FcγRIIIb, respectively. FcγR is a membraneprotein belonging to the immunoglobulin superfamily, FcγRII, FcγRIII,and FcγRIV have an a chain having an extracellular region containing twoimmunoglobulin-like domains, FcγRI has an a chain having anextracellular region containing three immunoglobulin-like domains, as aconstituting component, and the a chain is involved in the IgG bindingactivity. In addition, FcγRI and FcγRIII have a γ chain or ζ chain as aconstituting component which has a signal transduction function inassociation with the α chain (Annu. Rev. Immunol., 18, 709 (2000), Annu.Rev. Immunol., 19, 275 (2001)). FcγRIV has been described by Bruhns etal., Clin. Invest. Med., (Canada) 27:3D (2004).

To assess ADCC activity of an anti-CD19 antibody of interest, an invitro ADCC assay can be used, such as that described in U.S. Pat. No.5,500,362 or 5,821,337. The assay may also be performed using acommercially available kit, e.g. CytoTox 96® (Promega). Useful effectorcells for such assays include, but are not limited to peripheral bloodmononuclear cells (PBMC), Natural Killer (NK) cells, and NK cell lines.NK cell lines expressing a transgenic Fc receptor (e.g. CD16) andassociated signaling polypeptide (e.g. FCεRI-γ) may also serve aseffector cells (see, e.g. WO 2006/023148 A2 to Campbell). For example,the ability of any particular antibody to mediate lysis of the targetcell by complement activation and/or ADCC can be assayed. The cells ofinterest are grown and labeled in vitro; the antibody is added to thecell culture in combination with immune cells which may be activated bythe antigen antibody complexes; i.e., effector cells involved in theADCC response. The antibody can also be tested for complementactivation. In either case, cytolysis of the target cells is detected bythe release of label from the lysed cells. The extent of target celllysis may also be determined by detecting the release of cytoplasmicproteins (e.g. LDH) into the supernatant. In fact, antibodies can bescreened using the patient's own serum as a source of complement and/orimmune cells. The antibodies that are capable of mediating human ADCC inthe in vitro test can then be used therapeutically in that particularpatient. ADCC activity of the molecule of interest may also be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Moreover,techniques for modulating (i.e., increasing or decreasing) the level ofADCC, and optionally CDC activity, of an antibody are well-known in theart. See, e.g., U.S. Pat. No. 6,194,551. Antibodies of the presentinvention may be capable or may have been modified to have the abilityof inducing ADCC and/or CDC. Assays to determine ADCC function can bepracticed using human effector cells to assess human ADCC function. Suchassays may also include those intended to screen for antibodies thatinduce, mediate, enhance, block cell death by necrotic and/or apoptoticmechanisms. Such methods including assays utilizing viable dyes, methodsof detecting and analyzing caspases, and assays measuring DNA breaks canbe used to assess the apoptotic activity of cells cultured in vitro withan anti-CD19 antibody of interest.

For example, Annexin V or TdT-mediated dUTP nick-end labeling (TUNEL)assays can be carried out as described in Decker et al., Blood (USA)103:2718-2725 (2004) to detect apoptotic activity. The TUNEL assayinvolves culturing the cell of interest with fluorescein-labeled dUTPfor incorporation into DNA strand breaks. The cells are then processedfor analysis by flow cytometry. The Annexin V assay detects theappearance of phosphatidylserine (PS) on the outside of the plasmamembrane of apoptotic cells using a fluorescein-conjugated Annexin Vthat specifically recognizes the exposed PS molecules. Concurrently, aviable dye such as propidium iodide can be used to exclude lateapoptotic cells. The cells are stained with the labeled Annexin V andare analyzed by flow cytometry.

5.18.3. Immunoconjugates and Fusion Proteins

According to certain aspects of the invention, therapeutic agents ortoxins can be conjugated to chimerized, human, or humanized anti-CD19antibodies for use in compositions and methods of the invention. Incertain embodiments, these conjugates can be generated as fusionproteins. Examples of therapeutic agents and toxins include, but are notlimited to, members of the enediyne family of molecules, such ascalicheamicin and esperamicin. Chemical toxins can also be taken fromthe group consisting of duocarmycin (see, e.g., U.S. Pat. No. 5,703,080and U.S. Pat. No. 4,923,990), methotrexate, doxorubicin, melphalan,chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide,bleomycin and 5-fluorouracil. Examples of chemotherapeutic agents alsoinclude Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside(Ara-C), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan,Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine,Bleomycin, Etoposide, lfosfamide, Mitomycin C, Mitoxantrone,Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin,Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see,U.S. Pat. No. 4,675,187), Melphalan, and other related nitrogenmustards.

In certain embodiments, anti-CD19 antibodies are conjugated to acytostatic, cytotoxic or immunosuppressive agent wherein the cytotoxicagent is selected from the group consisting of an enediyne, alexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, amaytansinoid, and a vinca alkaloid. In certain, more specificembodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065,SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin,calicheamicin, maytansine, DM-1, auristatin E, AEB, AEVB, AEFP, MMAE(see, U.S. patent application Ser. No. 10/983,340; U.S. Pat. No.7,498,298), or netropsin.

In certain embodiments, the cytotoxic agent of an anti-CD19antibody-cytotoxic agent conjugate of the invention is an anti-tubulinagent. In specific embodiments, the cytotoxic agent is selected from thegroup consisting of a vinca alkaloid, a podophyllotoxin, a taxane, abaccatin derivative, a cryptophysin, a maytansinoid, a combretastatin,and a dolastatin. In other embodiments, the cytotoxic agent isvincristine, vinblastine, vindesine, vinorclbinc, VP-16, camptothccin,paclitaxcl, docctaxcl, cpithilonc A, cpithilonc B, nocodazole,coichicine, colcimid, estramustine, cemadotin, discodermolide,maytansine, DM-1, AEFP, auristatin E, AEB, AEVB, AEFP, MMAE oreleutherobin.

In specific embodiments, an anti-CD19 antibody is conjugated to thecytotoxic agent via a linker, wherein the linker is peptide linker. Inother embodiments, an anti-CD19 antibody is conjugated to the cytotoxicagent via a linker, wherein the linker is a val-cit linker, a phe-lyslinker, a hydrazone linker, or a disulfide linker.

In certain embodiments, the anti-CD19 antibody of an anti-CD19antibody-cytotoxic agent conjugate is conjugated to the cytotoxic agentvia a linker, wherein the linker is hydrolysable at a pH of less than5.5. In a specific embodiment the linker is hydrolyzable at a pH of lessthan 5.0.

In certain embodiments, the anti-CD19 antibody of an anti-CD19antibody-cytotoxic agent conjugate is conjugated to the cytotoxic agentvia a linker, wherein the linker is cleavable by a protease. In aspecific embodiment, the protease is a lysosomal protease. In otherembodiments, the protease is, inter alia, a membrane-associatedprotease, an intracellular protease, or an endosomal protease.

Other toxins that can be used in immunoconjugates of the inventioninclude poisonous lectins, plant toxins such as ricin, abrin, modeccin,botulina, and diphtheria toxins. Of course, combinations of the varioustoxins could also be coupled to one antibody molecule therebyaccommodating variable cytotoxicity. Illustrative of toxins which aresuitably employed in combination therapies of the invention are ricin,abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweedanti-viral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell, 47:641(1986), and Goldenberg et al., Cancer Journal for Clinicians, 44:43(1994). Enzymatically active toxins and fragments thereof which can beused include diphtheria A chain, non-binding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

Suitable toxins and chemotherapeutic agents are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and inGoodman And Gilman's The Pharmacological Basis of Therapeutics, 7th Ed.(MacMillan Publishing Co. 1985). Other suitable toxins and/orchemotherapeutic agents are known to those of skill in the art.

The present invention further encompasses antibodies (including antibodyfragments or variants thereof) comprising or conjugated to a radioactiveagent suitable for diagnostic purposes. Examples of suitable radioactivematerials include, but are not limited to, iodine (.sup.1211, .sup.123I,.sup.125I, .sup.131I), carbon (.sup.14C), sulfur (.sup.35S), tritium(.sup.3H), indium (.sup.111In, .sup.112In, .sup.113mIn, .sup.115mIn),technetium (.sup.99Tc, .sup.99mTc), thallium (.sup.201Ti), gallium(.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum (.sup.99Mo),xenon (.sup.135Xe), fluorine (.sup.18F), .sup.153Sm, .sup.177Lu,.sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y,.sup.47Sc, .sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, and.sup.97Ru.

Further, an anti-CD19 antibody of the invention (including an scFv orother molecule comprising, or alternatively consisting of, antibodyfragments or variants thereof), may be coupled or conjugated to aradioactive metal ion utilized for therapeutic purposes. Examples ofsuitable radioactive ions include, but are not limited to,alpha-emitters such as .sup.213Bi, or other radioisotopes such as.sup.103Pd, .sup.135Xe, .sup.131I, .sup.68Ge, .sup.57Co, .sup.65Zn,.sup.85Sr, .sup.32P, .sup.35S, .sup.90Y, .sup.153Sm, .sup.153Gd,.sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se, .sup.113 Sn, .sup.90Y,.sup.117Tin, .sup.186Re, .sup.188Re and .sup.166Ho. In specificembodiments, an antibody or fragment thereof is attached to macrocyclicchelators that chelate radiometal ions, including but not limited to,.sup.177Lu, .sup.90Y, .sup.166Ho, and .sup.153Sm, to polypeptides. Inspecific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclod-odecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the an antibody ofthe invention or fragment thereof via a linker molecule. Examples oflinker molecules useful for conjugating DOTA to a polypeptide arecommonly known in the art—see, for example, DeNardo et al., Clin CancerRes 4(10):2483-90, 1998; Peterson et al., Bioconjug Chem 10(4):553-7,1999; and Zimmerman et al., Nucl Med Biol 26(8):943-50, 1999 which arehereby incorporated by reference in their entirety.

An anti-CD19 antibody of the present invention may also be used in ADEPTby conjugating the antibody to a prodrug-activating enzyme whichconverts a prodrug (e.g., a peptidyl chemotherapeutic agent, see,WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378and U.S. Pat. No. 4,975,278. The enzyme component of the immunoconjugateuseful for ADEPT includes any enzyme capable of acting on a prodrug insuch a way so as to covert it into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with α-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Antibodies with enzymatic activity, also known in the art as“abzymes,” can be used as well to convert the prodrugs into free activedrugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme as desired to portions of a human affected by a B cellmalignancy.

Antibodies of this invention may be covalently bound to the enzymes bytechniques well-known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Fusionproteins comprising at least the antigen-binding region of an anti-CD19antibody linked to at least a functionally active portion of an enzymemay also be constructed using recombinant DNA techniques well-known inthe art (see, e.g., Neuberger et al., Nature, 312:604-608 (1984)).

Covalent modifications of an anti-CD19 antibody are included within thescope of this invention. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of an anti-CD19 antibody are introducedinto the molecule by reacting targeted amino acid residues of theantibody with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with a-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Similarly,iodo-reagents may also be used. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction canbe performed in 0.1 M sodium cacodylate at pH 6.0.

Lysyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues and/orε-amino-containing residues include imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, 0-methylisourea, 2,4-pentanedione, andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residuesgenerally requires that the reaction be performed in alkaline conditionsbecause of the high pKa of the guanidine functional group. Furthermore,these reagents may react with the ε-amino groups of lysine as well asthe arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to form0-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or 0-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

5.19. Chemotherapeutic Combinations

In other embodiments, an anti-CD19 mAb can be administered incombination with one or more additional chemotherapeutic agents. Forexample, “CVB” (1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and150-200 mg/m² carmustine) can be used in combination therapies of theinvention. CVB is a regimen used to treat non-Hodgkin's lymphoma (Pattiet al., Eur. J. Haematol., 51:18 (1993)). Other suitable combinationchemotherapeutic regimens are well-known to those of skill in the art.See, for example, Freedman et al., “Non-Hodgkin's Lymphomas,” in CancerMedicine, Volume 2, 3rd Edition, Holland et al. (eds.), pp. 2028-2068(Lea & Febiger 1993). As an illustration, first generationchemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma include C-MOPP (cyclophosphamide, vincristine,procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone, andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin, and leucovorin). Additional useful drugs include phenylbutyrate and brostatin-1

According to the invention, cancer or one or more symptoms thereof maybe prevented, treated, managed or ameliorated by the administration ofan anti-CD19 mAb in combination with the administration of one or moretherapies such as, but not limited to, chemotherapies, radiationtherapies, hormonal therapies, and/or biologicaltherapies/immunotherapies.

In a specific embodiment, methods of the invention encompass theadministration of one or more angiogenesis antagonists such as but notlimited to: Angiostatin (plasminogen fragment); antiangiogenicantithrombin III; Angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab;BMS-275291; cartilage-derived inhibitor (CDI); CAl; CD59 complementfragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagenXVIII fragment); Fibronectin fragment; Gro-beta; Halofuginone;Heparinases; Heparin hexasaccharide fragment; HMV833; Human chorionicgonadotropin (hCG); IM-862; Interferon alpha/beta/gamma; Interferoninducible protein (IP-10); Interleukin-12; Kringle 5 (plasminogenfragment); Marimastat; Metalloproteinase inhibitors (TIMPs);2-Methoxyestradiol; MMI 270 (CGS 27023A); MoAb IMC-1C11; Neovastat;NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Plasminogenactivator inhibitor; Platelet factor-4 (PF4); Prinomastat; Prolactin 16kD fragment; Proliferin-related protein (PRP); PTK 787/ZK 222594;Retinoids; Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248;Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1(TSP-1); TNP-470; Transforming growth factor-beta (TGF-b);Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD 6474;farnesyl transferase inhibitors (FTI); and bisphosphonates (such as butare not limited to, alendronate, clodronate, etidronate, ibandronate,pamidronate, risedronate, tiludronate, and zoledronate).

In a specific embodiment, methods of the invention encompass theadministration of one or more immunomodulatory agents, such as but notlimited to, chemotherapeutic agents and non-chemotherapeuticimmunomodulatory agents. Non-limiting examples of chemotherapeuticagents include methotrexate, cyclosporin A, leflunomide, cisplatin,ifosfamide, taxanes such as taxol and paclitaxol, topoisomerase Iinhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine,vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin,vinorelbine, temodal, cytochalasin B, gramicidin D, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin homologues, andcytoxan. Examples of non-chemotherapeutic immunomodulatory agentsinclude, but are not limited to, anti-T cell receptor antibodies (e.g.,anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (IDEC andSKB), mAB 4162W94, ORTHOCLONE® (muromonab-CD3) and OKTcdr4a(Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product DesignLabs), OKT3 (Johnson & Johnson), or RITUXAN™ (rituximab) (IDEC)),anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate),anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies,anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)),anti-CD52 antibodies (e.g., CAMPATH™ 1H (alemtuzumab) (Ilex)), anti-CD2antibodies (e.g., MEDI-507 (MedImmune, Inc., International PublicationNos. WO 02/098370 and WO 02/069904), anti-CD11a antibodies (e.g.,XANELIM™ (efalizumab) (Genentech)), and anti-B7 antibodies (e.g.,IDEC-114) (IDEC)); anti-cytokine receptor antibodies (e.g., anti-IFNreceptor antibodies, anti-IL-2 receptor antibodies (e.g., ZENAPAX™(daclizumab) (Protein Design Labs)), anti-IL-4 receptor antibodies,anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, andanti-IL-12 receptor antibodies), anti-cytokine antibodies (e.g.,anti-IFN antibodies, anti-TNF-α antibodies, anti-IL-1β antibodies,anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)),anti-IL-12 antibodies and anti-IL-23 antibodies)); CTLA4-immunoglobulin;LFA-3TIP (Biogen, International Publication No. WO 93/08656 and U.S.Pat. No. 6,162,432); soluble cytokine receptors (e.g., the extracellulardomain of a TNF-α receptor or a fragment thereof, the extracellulardomain of an IL-1β receptor or a fragment thereof, and the extracellulardomain of an IL-6 receptor or a fragment thereof); cytokines orfragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-23, TNF-α, TNF-β,interferon (IFN)-α, IFN-β, IFN-γ, and GM-CSF); and anti-cytokineantibodies (e.g., anti-IL-2 antibodies, anti-IL-4 antibodies, anti-IL-6antibodies, anti-IL-10 antibodies, anti-IL-12 antibodies, anti-IL-15antibodies, anti-TNF-α antibodies, and anti-IFN-γ antibodies), andantibodies that immunospecifically bind to tumor-associated antigens(e.g., HERCEPTIN™ (trastuzumab)). In certain embodiments, animmunomodulatory agent is an immunomodulatory agent other than achemotherapeutic agent. In other embodiments an immunomodulatory agentis an immunomodulatory agent other than a cytokine or hemapoietic suchas IL-1, IL-2, IL-4, IL-12, IL-15, TNF, IFN-α, IFN-β, IFN-γ, M-CSF,G-CSF, IL-3 or erythropoietin. In yet other embodiments, animmunomodulatory agent is an agent other than a chemotherapeutic agentand a cytokine or hemapoietic factor.

In a specific embodiment, methods of the invention encompass theadministration of one or more anti-inflammatory agents, such as but notlimited to, non-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingcric agents, andmethyl xanthincs. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™) fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

In another specific embodiment, methods of the invention encompass theadministration of one or more antiviral agents (e.g., amantadine,ribavirin, rimantadine, acyclovir, famciclovir, foscarnet, ganciclovir,trifluridine, vidarabine, didanosine, stavudine, zalcitabine,zidovudine, interferon), antibiotics (e.g., dactinomycin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC)),anti-emetics (e.g., alprazolam, dexamethoasone, domperidone, dronabinol,droperidol, granisetron, haloperidol, haloperidol, iorazepam,methylprednisolone, metoclopramide, nabilone, ondansetron,prochlorperazine), anti-fungal agents (e.g., amphotericin, clotrimazole,econazole, fluconazole, flucytosine, griseofulvin, itraconazole,ketoconazole, miconazole and nystatin), anti-parasite agents (e.g.,dehydroemetine, diloxanide furoate, emetine, mefloquine, melarsoprol,metronidazole, nifurtimox, paromomycin, pentabidine, pentamidineisethionate, primaquine, quinacrine, quinidine) or a combinationthereof.

Specific examples of anti-cancer agents that can be used in variousembodiments of the invention, including pharmaceutical compositions anddosage forms and kits, include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabinc;dacarbazinc; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1;interferon alpha-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofcnur; talisomycin; tccogalan sodium; tcgafur; tcloxantronchydrochloride; tcmoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altrctaminc; ambamustinc;amidox; amifostinc; aminolcvulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; cxemestanc; fadrozolc; fazarabinc; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacctatc; lanrcotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitor (suchas but not limited to, Lovastatin, Pravastatin, Fluvastatin, Statin,Simvastatin, and Atorvastatin); loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol;mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phcnazinomycin;phenylacetate; phosphatasc inhibitors; picibanil; pilocarpinchydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-tri amine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; R1I retinamide; rogletimide;rohitukinc; romurtidc; roquinimex; rubiginonc B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;Vitaxin®; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Additional anti-cancer drugs are 5-fluorouracil andleucovorin. These two agents may be useful when used in methodsemploying thalidomide and a topoisomerase inhibitor. In specificembodiments, an anti-cancer agent is not a chemotherapeutic agent.

In more particular embodiments, the present invention also comprises theadministration of an anti-CD19 mAb in combination with theadministration of one or more therapies such as, but not limited to,anti-cancer agents such as those disclosed in Table 1, for the treatmentof breast, ovary, melanoma, prostate, colon and lung cancers asdescribed above. When used in a combination therapy, the dosages and/orthe frequency of administration listed in Table 2 may be decreased.

TABLE 2 Anti-cancer agents Therapeutic AgentDose/Administration/Formulation doxorubicin Intravenous 60-75 mg/m² onDay 1 21 day intervals hydrochloride (Adriamycin RDF ® and AdriamycinPFS ® epirubicin Intravenous 100-120 mg/m² on Day 1 of 3-4 week cycleshydrochloride each cycle or (Ellence ™) divided equally and given onDays 1-8 of the cycle fluorousacil Intravenous How supplied: 5 mL and 10mL vials (containing 250 and 500 mg flourouracil respectively) docetaxelIntravenous 60-100 mg/m² over 1 hour Once every 3 weeks (Taxotere ®)paclitaxel Intravenous 175 mg/m² over 3 hours Every 3 weeks for(Taxol ®) 4 courses (administered sequentially to doxorubicin-containingcombination chemotherapy) tamoxifen citrate Oral 20-40 mg Daily(Nolvadex ®) (tablet) Dosages greater than 20 mg should be given individed doses (morning and evening) leucovorin intravenous How supplied:Dosage is unclear from calcium for or 350 mg vial text. PDR 3610injection intramuscular injection luprolide acetate single 1 mg (0.2 mLor 20 unit Once a day Lupron ®) subcutaneous mark) injection flutamideOral 50 mg 3 times a day at 8 hour (Eulexin ®) (capsule) (capsulescontain 125 mg intervals (total daily flutamide each) dosage 750 mg)nilutamide Oral 300 mg or 150 mg 300 mg once a day for 30 (Nilandron ®)(tablet) (tablets contain 50 or 150 mg days followed by 150 mgnilutamide each) once a day bicalutamide Oral 50 mg Once a day(Casodex ®) (tablet) (tablets contain 50 mg bicalutamide each)progesterone Injection USP in sesame oil 50 mg/mL ketoconazole Cream 2%cream applied once or (Nizoral ®) twice daily depending on symptomsprednisone Oral Initial dosage may vary from (tablet) 5 mg to 60 mg perday depending on the specific disease entity being treated. estramustineOral 14 mg/kg of body weight Daily given in 3 or 4 phosphate (capsule)(i.e. one 140 mg capsule for divided doses sodium each 10 kg or 22 lb ofbody (Emcyt ®) weight) etoposide or Intravenous 5 mL of 20 mg/mLsolution VP-16 (100 mg) dacarbazine Intravenous 2-4.5 mg/kg Once a dayfor 10 days. (DTIC-Dome ®) May be repeated at 4 week intervalspolifeprosan 20 wafer placed 8 wafers, each containing 7.7 mg withcarmustine in resection of carmustine, for a total implant (BCNU) cavityof 61.6 mg, if size and shape (nitrosourea) of resection cavity allows(Gliadel ®) cisplatin Injection [n/a in PDR 861] How supplied: solutionof 1 mg/mL in multi-dose vials of 50 mL and 100 mL mitomycin Injectionsupplied in 5 mg and 20 mg vials (containing 5 mg and 20 mg mitomycin)gemcitabine HCl Intravenous For NSCLC-2 schedules 4 week schedule-(Gemzar ®) have been investigated and Days 1, 8 and 15 of each theoptimum schedule has 28-day cycle. Cisplatin not been determinedintravenously at 100 mg/m² 4 week schedule- on day 1 after theadministration intravenously infusion of Gemzar. at 1000 mg/m² over 30 3week schedule- minutes on 3 week schedule- Days 1 and 8 of each 21Gemzar administered day cycle. Cisplatin at intravenously at 1250 mg/m²dosage of 100 mg/m² over 30 minutes administered intravenously afteradministration of Gemzar on day 1. carboplatin Intravenous Single agenttherapy: Every 4 weeks (Paraplatin ®) 360 mg/m² I.V. on day 1 (infusionlasting 15 minutes or longer) Other dosage calculations: Combinationtherapy with cyclophosphamide, Dose adjustment recommendations, Formuladosing, etc. ifosamide Intravenous 1.2 g/m² daily 5 consecutive days(Ifex ®) Repeat every 3 weeks or after recovery from hematologictoxicity topotecan Intravenous 1.5 mg/m² by intravenous 5 consecutivedays, hydrochloride infusion over 30 minutes starting on day 1 of 21 day(Hycamtin ®) daily course Bisphosphonates Intravenous 60 mg or 90 mgsingle Pamidronate or Oral infusion over 4-24 hours to Alendronate takewith correct hypercalcemia in Risedronate 6-8 oz cancer patients water.5 mg/d daily for 2 years and then 10 mg/d for 9 month to prevent orcontrol bone resorption. 5.0 mg to prevent or control bone resorption.Lovastatin Oral 10-80 mg/day in single or (Mevacor ™) two divided dose.

The invention also encompasses administration of an anti-CD19 mAb incombination with radiation therapy comprising the use of x-rays, gammarays and other sources of radiation to destroy the cancer cells. Inparticular embodiments, the radiation treatment is administered asexternal beam radiation or teletherapy wherein the radiation is directedfrom a remote source. In other embodiments, the radiation treatment isadministered as internal therapy or brachytherapy wherein a radiaoactivesource is placed inside the body close to cancer cells or a tumor mass.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (56^(th) ed., 2002).

5.20. Pharmaceutical Compositions

The invention also relates to immunotherapeutic compositions and methodsfor the treatment of B cell diseases and disorders in human subjects,such as, but not limited to, B cell malignancies, to immunotherapeuticcompositions and methods for the treatment and prevention of GVHD, graftrejection, and post-transplant lymphocyte proliferative disorder inhuman transplant recipients, and to immunotherapeutic compositions andmethods for the treatment of autoimmune diseases and disorders in humansubjects, using therapeutic antibodies that bind to the CD19 antigen andmay mediate human ADCC.

The present invention relates to pharmaceutical compositions comprisinghuman, humanized, or chimeric anti-CD19 antibodies of the IgG1 or IgG3human isotype. The present invention also relates to pharmaceuticalcompositions comprising human or humanized anti-CD19 antibodies of theIgG2 or IgG4 human isotype that may mediate human ADCC. In certainembodiments, the present invention also relates to pharmaceuticalcompositions comprising monoclonal human, humanized, or chimerizedanti-CD19 antibodies that can be produced by means known in the art.

Therapeutic formulations and regimens are described for treating humansubjects diagnosed with B cell malignancies that derive from B cells andtheir precursors, including but not limited to, acute lymphoblasticleukemias (ALL), Hodgkin's lymphomas, non-Hodgkin's lymphomas, B cellchronic lymphocytic leukemias (CLL), multiple myeloma, follicularlymphoma, mantle cell lymphoma, pro-lymphocytic leukemias, hairy cellleukemias, common acute lymphocytic leukemias and some Null-acutelymphoblastic leukemias.

In other particular embodiments, anti-CD19 antibodies may mediate ADCC,complement-dependent cellular cytoxicity, or apoptosis. Compositions andmethods of the present invention also have the advantage of targeting awider population of B cells than other B cell directed immunotherapies.For example, anti-CD19 antibodies of the present invention may beeffective to target bone marrow cells, circulating B cells, and mature,antibody-secreting B cells. Accordingly, methods and compositions of theinvention may be effective to reduce or deplete circulating B cells aswell as circulating immunoglobulin.

Accordingly, in one aspect, the invention provides compositions andmethods for the treatment and prevention of GVHD, graft rejection, andpost-transplantation lymphoproliferative disorder, which are associatedwith fewer and/or less severe complications than less-targetedtherapeutic agents and regimens. In one embodiment, compositions andmethods of the invention are used with lower doses of traditionaltherapeutic agents than would be possible in the absence of the methodsand compositions of the invention. In another embodiment, compositionsand methods of the invention obviate the need for a more severe form oftherapy, such as radiation therapy, high-dose chemotherapy, orsplenectomy.

In certain embodiments, anti-CD19 antibodies and compositions may beadministered to a transplant recipient patient prior to or followingtransplantation, alone or in combination with other therapeutic agentsor regimens for the treatment or prevention of GVHD and graft rejection.For example, anti-CD19 antibodies and compositions may be used todeplete alloantibodies from a transplant recipient prior to or followingtransplantation of an allogeneic graft. Anti-CD19 antibodies andcompositions may also be used to deplete antibody-producing cells fromthe graft ex vivo, prior to transplantation, or in the donor, or asprophylaxis against GVHD and graft rejection.

5.21. Pharmaceutical Formulations, Administration and Dosing

Pharmaceutical formulations of the invention contain as the activeingredient human, humanized, or chimeric anti-CD19 antibodies. Theformulations contain naked antibody, immunoconjugate, or fusion proteinin an amount effective for producing the desired response in a unit ofweight or volume suitable for administration to a human patient, and arepreferably sterile. The response can, for example, be measured bydetermining the physiological effects of the anti-CD19 antibodycomposition, such as, but not limited to, circulating B cell depletion,tissue B cell depletion, regression of a B cell malignancy, or decreaseof disease symptoms. Other assays will be known to one of ordinary skillin the art and can be employed for measuring the level of the response.

5.21.1. Pharmaceutical Formulations

An anti-CD19 antibody composition may be formulated with apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means one or more non-toxic materials that do not interferewith the effectiveness of the biological activity of the activeingredients. Such preparations may routinely contain salts, bufferingagents, preservatives, compatible carriers, and optionally othertherapeutic agents. Such pharmaceutically acceptable preparations mayalso routinely contain compatible solid or liquid fillers, diluents orencapsulating substances which are suitable for administration into ahuman. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic,malonic, succinic, and the like. Also, pharmaceutically acceptable saltscan be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts. The term “carrier” denotes anorganic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. Thecomponents of the pharmaceutical compositions also are capable of beingco-mingled with the antibodies of the present invention, and with eachother, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

According to certain aspects of the invention, anti-CD19 antibodycompositions can be prepared for storage by mixing the antibody orimmunoconjugate having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.(1999)), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes(e.g., Zn-protein complexes); and/or non-ionic surfactants such asTWEEN, PLURONICS™ or polyethylene glycol (PEG).

Anti-CD19 antibody compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

Anti-CD19 antibody compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, anti-CD19 antibody compositions areprepared by uniformly and intimately bringing the active compound intoassociation with a liquid carrier, a finely divided solid carrier, orboth, and then, if necessary, shaping the product.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of anti-CD19antibody, which is preferably isotonic with the blood of the recipient.This preparation may be formulated according to known methods usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administration canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. In certain embodiments, carrier formulation suitable forvarious routes of administration can be the same or similar to thatdescribed for RITUXAN™. See, Physicians' Desk Reference (MedicalEconomics Company, Inc., Montvale, N.J., 2005), pp. 958-960 and1354-1357, which is incorporated herein by reference in its entirety. Incertain embodiments of the invention, anti-CD19 antibody compositionsare formulated for intravenous administration with sodium chloride,sodium citrate dihydrate, polysorbate 80, and sterile water where the pHof the composition is adjusted to approximately 6.5. Those of skill inthe art are aware that intravenous injection provides a useful mode ofadministration due to the thoroughness of the circulation in rapidlydistributing antibodies. Intravenous administration, however, is subjectto limitation by a vascular barrier comprising endothelial cells of thevasculature and the subendothelial matrix. Still, the vascular barrieris a more notable problem for the uptake of therapeutic antibodies bysolid tumors. Lymphomas have relatively high blood flow rates,contributing to effective antibody delivery. Intralymphatic routes ofadministration, such as subcutaneous or intramuscular injection, or bycatheterization of lymphatic vessels, also provide a useful means oftreating B cell lymphomas. In certain embodiments, anti-CD19 antibodiesof compositions and methods of the invention are self-administeredsubcutaneously. In such embodiments, the composition is formulated as alyophilized drug or in a liquid buffer (e.g., PBS and/or citrate) atabout 50 mg/mL.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomcs, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration are typicallysterile. This is readily accomplished by filtration through sterilefiltration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing an anti-CD19 antibody, which matricesare in the form of shaped articles, e.g., films, or microcapsule.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devized for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulthydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions. In certain embodiments, the pharmaceutically acceptablecarriers used in compositions of the invention do not affect human ADCCor CDC.

Anti-CD19 antibody compositions disclosed herein may also be formulatedas immunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug (such as anti-CD19 antibodies disclosed herein) to ahuman. The components of the liposome are commonly arranged in a bilayerformation, similar to the lipid arrangement of biological membranes.Liposomes containing antibodies of the invention are prepared by methodsknown in the art, such as described in Epstein et al., Proc. Natl. Acad.Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA,77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomeswith enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. The antibody of the present invention can be conjugated to theliposomes as described in Martin et al., J. Biol. Chem., 257:286-288(1982) via a disulfide interchange reaction. A therapeutic agent canalso be contained within the liposome. See, Gabizon et al., J. NationalCancer Inst., (19)1484 (1989).

Some of the pharmaceutical formulations include, but are not limited to:

(a) a sterile, preservative-free liquid concentrate for intravenous(i.v.) administration of anti-CD19 antibody, supplied at a concentrationof 10 mg/ml in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.The product can be formulated for i.v. administration using sodiumchloride, sodium citrate dihydrate, polysorbate and sterile water forinjection. For example, the product can be formulated in 9.0 mg/mLsodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mLpolysorbate 80, and sterile water for injection. The pH is adjusted to6.5.

(b) A sterile, lyophilized powder in single-use glass vials forsubcutaneous (s.c.) injection. The product can be formulated withsucrose, L-histidine hydrochloride monohydrate, L-histidine andpolysorbate 20. For example, each single-use vial can contain 150 mganti-CD19 antibody, 123.2 mg sucrose, 6.8 mg L-histidine hydrochloridemonohydrate, 4.3 mg L-histidine, and 3 mg polysorbate 20. Reconstitutionof the single-use vial with 1.3 ml sterile water for injection yieldsapproximately 1.5 ml solution to deliver 125 mg per 1.25 ml (100 mg/ml)of antibody.

(c) A sterile, preservative-free lyophilized powder for intravenous(i.v.) administration. The product can be formulated with a-trehalosedihydrate, L-histidine HCl, histidine and polysorbate 20 USP. Forexample, each vial can contain 440 mg anti-CD19 antibody, 400 mgα,α-trehalose dihydrate, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, and1.8 mg polysorbate 20, USP. Reconstitution with 20 ml of bacteriostaticwater for injection (BWFI), USP, containing 1.1% benzyl alcohol as apreservative, yields a multi-dose solution containing 21 mg/ml antibodyat a pH of approximately 6.

(d) A sterile, lyophilized powder for intravenous infusion in which ananti-CD19 antibody is formulated with sucrose, polysorbate, monobasicsodium phosphate monohydrate, and dibasic sodium phosphate dihydrate.For example, each single-use vial can contain 100 mg antibody, 500 mgsucrose, 0.5 mg polysorbate 80, 2.2 mg monobasic sodium phosphatemonohydrate, and 6.1 mg dibasic sodium phosphate dihydrate. Nopreservatives are present. Following reconstitution with 10 ml sterilewater for injection, USP, the resulting pH is approximately 7.2.

(e) A sterile, preservative-free solution for subcutaneousadministration supplied in a single-use, 1 ml pre-filled syringe. Theproduct can be formulated with sodium chloride, monobasic sodiumphosphate dihydrate, dibasic sodium phosphate dihydrate, sodium citrate,citric acid monohydrate, mannitol, polysorbate 80 and water forinjection, USP. Sodium hydroxide may be added to adjust pH to about 5.2.

For example, each syringe can be formulated to deliver 0.8 ml (40 mg) ofdrug product. Each 0.8 ml contains 40 mg anti-CD19 antibody, 4.93 mgsodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mgdibasic sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 citricacid monohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80 and water forinjection, USP.

(f) A sterile, preservative-free, lyophilized powder contained in asingle-use vial that is reconstituted with sterile water for injection(SWFI), USP, and administered as a subcutaneous (s.c.) injection. Theproduct can be formulated with sucrose, histidine hydrochloridemonohydrate, L-histidine, and polysorbate. For example, a 75 mg vial cancontain 129.6 mg or 112.5 mg of an anti-CD19 antibody, 93.1 mg sucrose,1.8 mg L-histidine hydrochloride monohydrate, 1.2 mg L-histidine, and0.3 mg polysorbate 20, and is designed to deliver 75 mg of the antibodyin 0.6 ml after reconstitution with 0.9 ml SWFI, USP. A 150 mg vial cancontain 202.5 mg or 175 mg anti-CD19 antibody, 145.5 mg sucrose, 2.8 mgL-histidine hydrochloride monohydrate, 1.8 mg L-histidine, and 0.5 mgpolysorbate 20, and is designed to deliver 150 mg of the antibody in 1.2ml after reconstitution with 1.4 ml SWFI, USP.

(g) A sterile, hyophilized product for reconstitution with sterile waterfor injection. The product can be formulated as single-use vials forintramuscular (IM) injection using mannitol, histidine and glycine. Forexample, each single-use vial can contain 100 mg anti-CD19 antibody,67.5 mg of mannitol, 8.7 mg histidine and 0.3 mg glycine, and isdesigned to deliver 100 mg antibody in 1.0 ml when reconstituted with1.0 ml sterile water for injection. As another example, each single-usevial can contain 50 mg anti-CD19 antibody, 40.5 mg mannitol, 5.2 mghistidine and 0.2 mg glycine, and is designed to deliver 50 mg ofantibody when reconstituted with 0.6 ml sterile water for injection.

(h) A sterile, preservative-free solution for intramuscular (IM)injection, supplied at a concentration of 100 mg/ml. The product can beformulated in single-use vials with histidine, glycine, and sterilewater for injection. For example, each single-use vial can be formulatedwith 100 mg antibody, 4.7 mg histidine, and 0.1 mg glycine in a volumeof 1.2 ml designed to deliver 100 mg of antibody in 1 ml. As anotherexample, each single-use vial can be formulated with 50 mg antibody, 2.7mg histidine and 0.08 mg glycine in a volume of 0.7 ml or 0.5 mldesigned to deliver 50 mg of antibody in 0.5 ml.

In certain embodiments, a pharmaceutical composition of the invention isstable at 4° C. In certain embodiments, a pharmaceutical composition ofthe invention is stable at room temperature.

5.21.2. Antibody Half-Life

In certain embodiments, the half-life of an anti-CD19 antibody ofcompositions and methods of the invention is at least about 4 to 7 days.In certain embodiments, the mean half-life of an anti-CD19 antibody ofcompositions and methods of the invention is at least about 2 to 5 days,3 to 6 days, 4 to 7 days, 5 to 8 days, 6 to 9 days, 7 to 10 days, 8 to11 days, 8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to18, 15 to 19, or 16 to 20 days. In other embodiments, the mean half-lifeof an anti-CD19 antibody of compositions and methods of the invention isat least about 17 to 21 days, 18 to 22 days, 19 to 23 days, 20 to 24days, 21 to 25, days, 22 to 26 days, 23 to 27 days, 24 to 28 days, 25 to29 days, or 26 to 30 days. In still further embodiments the half-life ofan anti-CD19 antibody of compositions and methods of the invention canbe up to about 50 days. In certain embodiments, the half-lives ofantibodies of compositions and methods of the invention can be prolongedby methods known in the art. Such prolongation can in turn reduce theamount and/or frequency of dosing of the antibody compositions.Antibodies with improved in vivo half-lives and methods for preparingthem are disclosed in U.S. Pat. No. 6,277,375; and InternationalPublication Nos. WO 98/23289 and WO 97/3461.

The serum circulation of anti-CD19 antibodies in vivo may also beprolonged by attaching inert polymer molecules such as high molecularweight polyethyleneglycol (PEG) to the antibodies with or without amultifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of the antibodies or via epsilon-aminogroups present on lysyl residues. Linear or branched polymerderivatization that results in minimal loss of biological activity willbe used. The degree of conjugation can be closely monitored by SDS-PAGEand mass spectrometry to ensure proper conjugation of PEG molecules tothe antibodies. Unreacted PEG can be separated from antibody-PEGconjugates by size-exclusion or by ion-exchange chromatography.PEG-derivatized antibodies can be tested for binding activity as well asfor in vivo efficacy using methods known to those of skill in the art,for example, by immunoassays described herein.

Further, the antibodies of compositions and methods of the invention canbe conjugated to albumin in order to make the antibody more stable invivo or have a longer half-life in vivo. The techniques are well knownin the art, see, e.g., International Publication Nos. WO 93/15199, WO93/15200, and WO 01/77137; and European Patent No. EP 413, 622, all ofwhich are incorporated herein by reference.

5.21.3. Administration and Dosing

Administration of compositions of the invention to a human patient canbe by any route, including but not limited to intravenous, intradermal,transdermal, subcutaneous, intramuscular, inhalation (e.g., via anaerosol), buccal (e.g., sub-lingual), topical (i.e., both skin andmucosal surfaces, including airway surfaces), intrathecal,intraarticular, intraplural, intracerebral, intra-arterial,intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginaladministration, by perfusion through a regional catheter, or by directintralesional injection. In one embodiment, compositions of theinvention are administered by intravenous push or intravenous infusiongiven over defined period (e.g., 0.5 to 2 hours). Compositions of theinvention can be delivered by peristaltic means or in the form of adepot, although the most suitable route in any given case will depend,as is well known in the art, on such factors as the species, age, genderand overall condition of the subject, the nature and severity of thecondition being treated and/or on the nature of the particularcomposition (i.e., dosage, formulation) that is being administered. Inparticular embodiments, the route of administration is via bolus orcontinuous infusion over a period of time, once or twice a week. Inother particular embodiments, the route of administration is bysubcutaneous injection, optionally once or twice weekly. In oneembodiment, compositions, and/or methods of the invention areadministered on an outpatient basis.

In certain embodiments, the dose of a composition comprising anti-CD19antibody is measured in units of mg/kg of patient body weight. In otherembodiments, the dose of a composition comprising anti-CD19 antibody ismeasured in units of mg/kg of patient lean body weight (i.e., bodyweight minus body fat content). In yet other embodiments, the dose of acomposition comprising anti-CD19 antibody is measured in units of mg/m²of patient body surface area. In yet other embodiments, the dose of acomposition comprising anti-CD19 antibody is measured in units of mg perdose administered to a patient. Any measurement of dose can be used inconjunction with compositions and methods of the invention and dosageunits can be converted by means standard in the art.

Those skilled in the art will appreciate that dosages can be selectedbased on a number of factors including the age, sex, species andcondition of the subject (e.g., stage of B cell malignancy), the desireddegree of cellular depletion, the disease to be treated and/or theparticular antibody or antigen-binding fragment being used and can bedetermined by one of skill in the art. For example, effective amounts ofcompositions of the invention may be extrapolated from dose-responsecurves derived in vitro test systems or from animal model (e.g., thecotton rat or monkey) test systems. Models and methods for evaluation ofthe effects of antibodies are known in the art (Wooldridge et al.,Blood, 89(8): 2994-2998 (1997)), incorporated by reference herein in itsentirety). In certain embodiments, for particular B cell malignancies,therapeutic regimens standard in the art for antibody therapy can beused with compositions and methods of the invention.

Examples of dosing regimens that can be used in methods of the inventioninclude, but are not limited to, daily, three times weekly(intermittent), weekly, or every 14 days. In certain embodiments, dosingregimens include, but are not limited to, monthly dosing or dosing every6-8 weeks.

Those skilled in the art will appreciate that dosages are generallyhigher and/or frequency of administration greater for initial treatmentas compared with maintenance regimens.

In some embodiments of the invention, anti-CD19 antibodies bind to Bcells and may result in efficient (i.e., at low dosage) depletion of Bcells (as described herein). Higher degrees of binding may be achievedwhere the density of human CD19 on the surface of a patient's B cells ishigh. In certain embodiments, dosages of the antibody (optionally in apharmaceutically acceptable carrier as part of a pharmaceuticalcomposition) are at least about 0.0005, 0.001, 0.05, 0.075, 0.1, 0.25,0.375, 0.5, 1, 2.5, 5, 10, 20, 37.5, or 50 mg/m2 and/or less than about500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175,150, 125, 100, 75, 60, 50, 37.5, 20, 15, 10, 5, 2.5, 1, 0.5, 0.375, 0.1,0.075 or 0.01 mg/m². In certain embodiments, the dosage is between about0.0005 to about 200 mg/m², between about 0.001 and 150 mg/m², betweenabout 0.075 and 125 mg/m², between about 0.375 and 100 mg/m², betweenabout 2.5 and 75 mg/m², between about 10 and 75 mg/m², and between about20 and 50 mg/m². In related embodiments, the dosage of anti-CD19antibody used is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5 mg/kg of body weight of apatient. In certain embodiments, the dose of naked anti-CD19 antibodyused is at least about 1 to 10, 5 to 15, 10 to 20, or 15 to 25 mg/kg ofbody weight of a patient. In certain embodiments, the dose of anti-CD19antibody used is at least about 1 to 20, 3 to 15, or 5 to 10 mg/kg ofbody weight of a patient. In other embodiments, the dose of anti-CD19antibody used is at least about 5, 6, 7, 8, 9, or 10 mg/kg of bodyweight of a patient. In certain embodiments, a single dosage unit of theantibody (optionally in a pharmaceutically acceptable carrier as part ofa pharmaceutical composition) can be at least about 0.5, 1, 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, or 250micrograms/m². In other embodiments, dose is up to 1 g per single dosageunit.

All of the above doses are exemplary and can be used in conjunction withcompositions and methods of the invention, however where an anti-CD19antibody is used in conjunction with a toxin or radiotherapeutic agentthe lower doses described above may be preferred. In certainembodiments, where the patient has low levels of CD19 density, the lowerdoses described above may be preferred.

In certain embodiments of the invention where chimeric anti-CD19antibodies are used, the dose or amount of the chimeric antibody isgreater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16mg/kg of patient body weight. In other embodiments of the inventionwhere chimeric anti-CD19 antibodies are used, the dose or amount of thechimeric antibody is less than about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2, or 0.1 mg/kg of patient body weight.

In some embodiments of methods of this invention, antibodies and/orcompositions of this invention can be administered at a dose lower thanabout 375 mg/m²; at a dose lower than about 37.5 mg/m²; at a dose lowerthan about 0.375 mg/m²; and/or at a dose between about 0.075 mg/m² andabout 125 mg/m². In certain embodiments of methods of the invention,dosage regimens comprise low doses, administered at repeated intervals.For example, in one embodiment, compositions of the invention can beadministered at a dose lower than about 375 mg/m² at intervals ofapproximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 days.

The specified dosage can result in B cell depletion in the human treatedusing compositions and methods of the invention for a period of at leastabout 1, 2, 3, 5, 7, 10, 14, 20, 30, 45, 60, 75, 90, 120, 150 or 180days or longer. In certain embodiments, pre-B cells (not expressingsurface immunoglobulin) are depleted. In certain embodiments, mature Bcells (expressing surface immunoglobluin) are depleted. In otherembodiments, all non-malignant types of B cells can exhibit depletion.Any of these types of B cells can be used to measure B cell depletion. Bcell depletion can be measured in bodily fluids such as blood serum, orin tissues such as bone marrow. In certain embodiments of methods of theinvention, B cells are depleted by at least 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% in comparison to B cell levels in the patient beingtreated before use of compositions and methods of the invention. Inother embodiments of methods of the invention, B cells are depleted byat least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in comparison totypical standard B cell levels for humans. In related embodiments, thetypical standard B cell levels for humans are determined using patientscomparable to the patient being treated with respect to age, sex,weight, and other factors.

In certain embodiments of the invention, a dosage of about 125 mg/m² orless of an antibody or antigen-binding fragment results in B celldepletion for a period of at least about 7, 14, 21, 30, 45, 60, 90, 120,150, or 200 days. In another representative embodiment, a dosage ofabout 37.5 mg/m² or less depletes B cells for a period of at least about7, 14, 21, 30, 45, 60, 90, 120, 150, or 200 days. In still otherembodiments, a dosage of about 0.375 mg/m² or less results in depletionof B cells for at least about 7, 14, 21, 30, 45 or 60 days. In anotherembodiment, a dosage of about 0.075 mg/m² or less results in depletionof B cells for a period of at least about 7, 14, 21, 30, 45, 60, 90,120, 150, or 200 days. In yet other embodiments, a dosage of about 0.01mg/m², 0.005 mg/m² or even 0.001 mg/m² or less results in depletion of Bcells for at least about 3, 5, 7, 10, 14, 21, 30, 45, 60, 90, 120, 150,or 200 days. According to these embodiments, the dosage can beadministered by any suitable route, but is optionally administered by asubcutaneous route.

As another aspect, the invention provides the discovery that B celldepletion and/or treatment of B cell disorders can be achieved at lowerdosages of antibody or antibody fragments than employed in currentlyavailable methods. Thus, in another embodiment, the invention provides amethod of depleting B cells and/or treating a B cell disorder,comprising administering to a human, an effective amount of an antibodythat specifically binds to CD19, wherein a dosage of about 500, 475,450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125,100, 75, 60, 50, 37.5, 20, 10, 5, 2.5, 1, 0.5, 0.375, 0.25, 0.1, 0.075,0.05, 0.001, 0.0005 mg/m² or less results in a depletion of B cells(circulating and/or tissue B cells) of 25%, 35%, 50%, 60%, 75%, 80%,85%, 90%, 95%, 98% or more for a period at least about 3, 5, 7, 10, 14,21, 30, 45, 60, 75, 90, 120, 150, 180, or 200 days or longer. Inrepresentative embodiments, a dosage of about 125 mg/m² or 75 mg/m² orless results in at least about 50%, 75%, 85% or 90% depletion of B cellsfor at least about 7, 14, 21, 30, 60, 75, 90, 120, 150 or 180 days. Inother embodiments, a dosage of about 50, 37.5 or 10 mg/m² results in atleast about a 50%, 75%, 85% or 90% depletion of B cells for at leastabout 7, 14, 21, 30, 60, 75, 90, 120 or 180 days. In still otherembodiments, a dosage of about 0.375 or 0.1 mg/m² results in at leastabout a 50%, 75%, 85% or 90% depletion of B cells for at least about 7,14, 21, 30, 60, 75 or 90 days. In further embodiments, a dosage of about0.075, 0.01, 0.001, or 0.0005 mg/m² results in at least about a 50%,75%, 85% or 90% depletion of B cells for at least about 7, 14, 21, 30 or60 days.

In certain embodiments of the invention, the dose can be escalated orreduced to maintain a constant dose in the blood or in a tissue, suchas, but not limited to, bone marrow. In related embodiments, the dose isescalated or reduced by about 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, and 95% in order to maintain a desired level of anantibody of compositions and methods of the invention.

In certain embodiments, the dosage can be adjusted and/or the infusionrate can be reduced based on patient's immunogenic response tocompositions and methods of the invention.

According to one aspect of methods of the invention, a loading dose ofan anti-CD19 antibody and/or composition of the invention can beadministered first followed by a maintenance dose until the B cellmalignancy being treated progresses or followed by a defined treatmentcourse (e.g., CAMPATH™ (alemtuzumab), MYLOTARG™ (gemtuzumab ozogamicin),or RITUXAN™ (rituximab), the latter of which allow patients to betreated for a defined number of doses that has increased as additionaldata have been generated).

According to another aspect of methods of the invention, a patient maybe pretreated with compositions and methods of the invention to detect,minimize immunogenic response, or minimize adverse effects ofcompositions and methods of the invention.

5.21.4. Toxicity Testing

The tolerance, toxicity and/or efficacy of the compositions and/ortreatment regimens of the present invention can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation), the ED50 (the dose therapeutically effective in 50% of thepopulation), and IC50 (the dose effective to achieve a 50% inhibition).In one embodiment, the dose is a dose effective to achieve at least a60%, 70%, 80%, 90%, 95%, or 99% depletion of circulating B cells orcirculating immunogloblulin, or both. The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Therapies that exhibit large therapeutic indicesmay be preferred. While therapies that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchagents to CD19-expressing cells in order to minimize potential damage toCD19 negative cells and, thereby, reduce side effects.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosages of the compositions and/ortreatment regimens for use in humans. The dosage of such agents may liewithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For any therapy used in methods of the invention, a therapeuticallyeffective dose can be estimated by appropriate animal models. Dependingon the species of the animal model, the dose can be scaled for human useaccording to art-accepted formulas, for example, as provided byFreireich et al., Quantitative comparison of toxicity of anticanceragents in mouse, rat, monkey, dog, and human, Cancer ChemotherapyReports, NCI 1966 40:219-244. Data obtained from cell culture assays canbe useful for predicting potential toxicity. Animal studies can be usedto formulate a specific dose to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Plasma drug levels may bemeasured, for example, by high performance liquid chromatography, ELISA,or by cell based assays.

5.22. Patient Diagnosis, Staging and Therapeutic Regimens Oncology

According to certain aspects of the invention, the treatment regimen anddose used with compositions and methods of the invention is chosen basedon a number of factors including, but not limited to, the stage of the Bcell disease or disorder being treated. Appropriate treatment regimenscan be determined by one of skill in the art for particular stages of aB cell disease or disorder in a patient or patient population. Doseresponse curves can be generated using standard protocols in the art inorder to determine the effective amount of compositions of the inventionfor treating patients having different stages of a B cell disease ordisorder. In general, patients having more advanced stages of a B celldisease or disorder will require higher doses and/or more frequent doseswhich may be administered over longer periods of time in comparison topatients having an early stage B cell disease or disorder.

Anti-CD19 antibodies, compositions and methods of the invention may bepracticed to treat B cell diseases, including B cell malignancies. Theterm “B cell malignancy” includes any malignancy that is derived from acell of the B cell lineage. Exemplary B cell malignancies include, butare not limited to: B cell subtype non-Hodgkin's lymphoma (NHL)including low grade/follicular NHL, small lymphocytic (SL) NHL,intermediate grade/follicular NHL, intermediate grade diffuse NHL, highgrade immunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL; mantle-cell lymphoma, and bulky disease NHL;Burkitt's lymphoma; multiple myeloma; pre-B acute lymphoblastic leukemiaand other malignancies that derive from early B cell precursors; commonacute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL)including immunoglobulin-mutated CLL and immunoglobulin-unmutated CLL;hairy cell leukemia; Null-acute lymphoblastic leukemia; Waldenstrom'sMacroglobulinemia; diffuse large B cell lymphoma (DLBCL) includinggerminal center B cell-like (GCB) DLBCL, activated B cell-like (ABC)DLBCL, and type 3 DLBCL; pro-lymphocytic leukemia; light chain disease;plasmacytoma; osteosclerotic myeloma; plasma cell leukemia; monoclonalgammopathy of undetermined significance (MGUS); smoldering multiplemyeloma (SMM); indolent multiple myeloma (IMM); Hodgkin's lymphomaincluding classical and nodular lymphocyte pre-dominant type;lymphoplasmacytic lymphoma (LPL); and marginal-zone lymphoma includinggastric mucosal-associated lymphoid tissue (MALT) lymphoma.

In a further embodiment the invention can be employed to treat mature Bcell malignancies (i.e., express Ig on the cell surface) including butnot limited to follicular lymphoma, mantle-cell lymphoma, Burkitt'slymphoma, multiple myeloma, diffuse large B-cell lymphoma (DLBCL)including germinal center B cell-like (GCB) DLBCL, activated B cell-like(ABC) DLBCL, and type 3 DLBCL, Hodgkin's lymphoma including classicaland nodular lymphocyte pre-dominant type, lymphoplasmacytic lymphoma(LPL), marginal-zone lymphoma including gastric mucosal-associatedlymphoid tissue (MALT) lymphoma, and chronic lymphocytic leukemia (CLL)including immunoglobulin-mutated CLL and immunoglobulin-unmutated CLL.

Further, CD19 is expressed earlier in B cell development than, forexample, CD20, and is therefore particularly suited for treating pre-Bcell and immature B cell malignancies (i.e., do not express Ig on thecell surface), for example, in the bone marrow. Illustrative pre-B celland immature B cell malignancies include, but are not limited to, acutelymphoblastic leukemia.

In other particular embodiments, the invention can be practiced to treatextranodal tumors.

5.22.1. Diagnosis and Staging of B Cell Malignancies

The progression of cancer, such as a B cell disease or disorder capableof tumor formation (e.g., non-Hodgkin lymphoma, diffuse large B celllymphoma, follicular lymphoma, and Burkitt lymphoma) is typicallycharacterized by the degree to which the cancer has spread through thebody and is often broken into the following four stages which areprognostic of outcome. Stage I: The cancer is localized to a particulartissue and has not spread to the lymph nodes. Stage II: The cancer hasspread to the nearby lymph nodes, i.e., metastasis. Stage III: Thecancer is found in the lymph nodes in regions of the body away from thetissue of origin and may comprise a mass or multiple tumors as opposedto one. Stage IV: The cancer has spread to a distant part of the body.The stage of a cancer can be determined by clinical observations andtesting methods that are well known to those of skill in the art. Thestages of cancer described above are traditionally used in conjunctionwith clinical diagnosis of cancers characterized by tumor formation, andcan be used in conjunction with the compositions and methods of thepresent invention to treat B cell diseases and disorders. Typicallyearly stage disease means that the disease remains localized to aportion of a patient's body or has not metastasized.

With respect to non-tumor forming B cell diseases and disorders such as,but not limited to, multiple myeloma, the criteria for determining thestage of disease differs. The Durie-Salmon Staging System has beenwidely used. In this staging system, clinical stage of disease (stage I,II, or III) is based on several measurements, including levels of Mprotein, the number of lytic bone lesions, hemoglobin values, and serumcalcium levels. Stages are further divided according to renal (kidney)function (classified as A or B). According to the Durie-Salmon StagingSystem Stage I (low cell mass) is characterized by all of the following:Hemoglobin value >10 g/dL; Serum calcium value normal or <12 mg/dL; Bonex-ray, normal bone structure (scale 0) or solitary bone plasmacytomaonly; and Low M-component production rate: IgG value <5 g/dL, IgA value<3 g/d, Bence Jones protein <4 g/24 h. Stage I patients typically haveno related organ or tissue impairment or symptoms. Stage II(intermediate cell mass) is characterized by fitting neither stage I norstage III. Stage III (high cell mass) is characterized by one or more ofthe following: Hemoglobin value <8.5 g/dL; Serum calcium value >12mg/dL; Advanced lytic bone lesions (scale 3); High M-componentproduction rate: IgG value >7 g/dL, IgA value >5 g/dL, Bence Jonesprotein >12 g/24 h Subclassification (either A or B), where A isRelatively normal renal function (serum creatinine value <2.0 mg/dL) andB is Abnormal renal function (serum creatinine value >2.0 mg/dL).

Another staging system for myeloma is the International Staging System(ISS) for myeloma. This system can more effectively discriminate betweenstaging groups and is based on easily measured serum levels of beta2-microglobulin (β2-M) and albumin. According to the ISS for myeloma,Stage I is characterized by β2-M<3.5 and Albumin >3.5, Stage II ischaracterized by β2-M<3.5 and albumin <3.5 or β2-M 3.5-5.5, and StageIII is characterized by β2-M>5.5 (Multiple Myeloma Research Foundation,New Canaan, Conn.).

The stage of a B cell malignancy in a patient is a clinicaldetermination. As indicated above, with respect to solid tumors, thespread, location, and number of tumors are the primary factors in theclinical determination of stage. Determination of stage in patients withnon-tumor forming B cell malignancies can be more complex requiringserum level measurements as described above.

The descriptions of stages of B cell diseases and disorders above arenot limiting. Other characteristics known in the art for the diagnosisof B cell diseases and disorders can be used as criteria for patients todetermine stages of B cell diseases or disorders.

5.22.2. Clinical Criteria for Diagnosing B Cell Malignancies

Diagnostic criteria for different B cell malignancies are known in theart. Historically, diagnosis is typically based on a combination ofmicroscopic appearance and immunophenotype. More recently, moleculartechniques such as gene-expression profiling have been applied todevelop molecular definitions of B cell malignancies (see, e.g., Shafferet al., Nature 2:920-932 (2002)). Exemplary methods for clinicaldiagnosis of particular B cell malignancies are provided below. Othersuitable methods will be apparent to those skilled in the art.

5.22.2.1. Follicular NHL

In general, most NHL (with the exception of mantle-cell lymphoma) havehighly mutated immunoglobulin genes that appear to be the result ofsomatic hypermutation (SHM). The most common genetic abnormalities inNHL are translocations and mutations of the BCL6 gene.

Follicular NHL is often an indolent B cell lymphoma with a folliculargrowth pattern. It is the second most common lymphoma in the UnitedStates and Western Europe. The median age at which this disease presentsis 60 years and there is a slight female predominance. Painlesslymphadenopathy is the most common symptom. Tests often indicateinvolvement of the blood marrow and sometimes the peripheral blood.Follicular NHL is divided into cytologic grades based on the proportionof large cells in the follicle with the grades forming a continuum fromfollicular small cleaved-cell to large-cell predominance. (See, S.Freedman, et al., Follicular Lymphoma, pp. 367-388, In Non-Hodgkin'sLymphomas, P. Mauch et al., eds., Lippincott Williams & Wilkins,Philadelphia, Pa. (2004); T. Lister et al., Follicular Lymphoma, pp.309-324, In Malignant Lymphoma, B. Hancock et al., eds., OxfordUniversity Press, New York, N.Y. (2000)).

Most follicular NHL is characterized by a translocation betweenchromosomes 14 and 18 resulting in overexpression of BCL2. FollicularNHL is also characterized by both SHM and ongoing SHM and a geneexpression profile similar to germinal center (GC) B cells (see, e.g.,Shaffer et al., Nature 2:920-932 (2002)), which are the putative cellsof origin for this malignancy. Heavy- and light chain rearrangements aretypical. The tumor cells of this disease express monoclonal surfaceimmunoglobulin with most expressing IgM. Nearly all follicular NHL tumorcells express the antigens CD19, CD20, CD22, CD79a, CD21, CD35 and CD10but lack expression of CD5 and CD43. Paratrabecular infiltration withsmall cleaved cells is observed in the bone marrow. (See, S. Freedman etal., Follicular Lymphoma, pp. 367-388, In Non-Hodgkin's Lymphomas, P.Mauch et al., eds., Lippincott Williams & Wilkins, Philadelphia, Pa.(2004); T. Lister et al., Follicular Lymphoma, pp. 309-324, In MalignantLymphoma, B. Hancock et al., eds., Oxford University Press, New York,N.Y. (2000)).

Diagnosis of follicular NHL generally relies on biopsy of an excisednode in order to evaluate tissue architecture and cytological features.Fine-needle aspirations are usually not adequate since this procedure isless likely to provide tissue that can be evaluated and it fails toprovide enough tissue for additional tests. Bilateral bone marrowbiopsies are also indicated since involvement can be patchy. Additionaldiagnostic procedures include chest x-rays, chest, abdomen, neck andpelvis computed tomography (CT) scans, complete blood count, andchemistry profile. Flow cytometry and immunohistochemistry can be usedto distinguish between follicular NHL and other mature B cell lymphomas.(See, S. Freedman et al., Follicular Lymphoma, pp. 367-388, InNon-Hodgkin's Lymphomas, P. Mauch et al., eds., Lippincott Williams &Wilkins, Philadelphia, Pa. (2004); T. Lister et al., FollicularLymphoma, pp. 309-324, In Malignant Lymphoma, B. Hancock et al., eds.,Oxford University Press, New York, N.Y. (2000)).

5.22.2.2. Mantle-Cell Lymphoma

Mantle-cell lymphoma localizes to the mantle region of secondaryfollicles and is characterized by a nodular and/or diffuse growthpattern. Mantle-cell lymphoma patients have median age of 60-65 yearswith the disease affecting predominantly males. For diagnostic purposes,the usual presenting feature is a generalized lymphadenopathy.Additionally, the spleen is often enlarged. This B cell lymphoma isassociated with a t(11;14) between the IgH locus and cyclin D1 gene,which results in overexpression of cyclin D1. More than 50% of casesshow additional chromosomal abnormalities. Mantle-cell lymphoma istypically not characterized by SHM. (See, W. Hiddemann et al., MantleCell Lymphoma, pp. 461-476, In Non-Hodgkin's Lymphomas, P. Mauch et al.,eds., Lippincott Williams & Wilkins, Philadelphia, Pa. (2004); D.Weisenburger et al., Mantle Cell Lymphoma, pp. 28-41, In MalignantLymphoma, B. Hancock et al., eds., Oxford University Press, New York,N.Y. (2000)).

Immunophenotyping (flow cytometry or frozen section)immunohistochemistry of mantle cell lymphoma cells shows them to nearlyalways be monoclonal, bearing surface IgM. Mantle cell lymphoma cellshave also been noted to bear surface IgD. The cells express the antigensCD19, CD20, CD22 and CD24, but not CD23. They also express surfaceantigens CD5 but not for CD10, distinguishing them from true folliclecenter-cell lymphomas which are almost always CD5 negative. Frequently,extranodal involvement is found including bone marrow infiltration andtumors of the liver and gastrointestinal tract. Mild anemia and leukemicexpression is not uncommon with mantle-cell lymphoma. (See, A. Lal etal., Role of Fine Needle Aspiration in Lymphoma, pp. 181-220, In W. Finnet al., eds., Hematopathology in Oncology, Kluwer Academic Publishers,Norwell, Mass. (2004); W. Hiddemann et al., Mantle Cell Lymphoma, pp.461-476, In Non-Hodgkin's Lymphomas, P. Mauch et al., eds., LippincottWilliams & Wilkins, Philadelphia, Pa. (2004)).

Diagnosis of mantle-cell lymphoma involves examination of the peripheralblood as well as bone marrow and lymph node biopsies. In addition,cytogenetic studies and immunophenotyping are useful in differentialdiagnosis. (See, W. Hiddemann, et al., Mantle Cell Lymphoma pp. 461-476,In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds., Lippincott Williams& Wilkins, Philadelphia, Pa. (2004); D. Weisenburger, et al., MantleCell Lymphoma, pp. 28-41, In Malignant Lymphoma, B. Hancock, et al.,eds., Oxford University Press, New York, N.Y. (2000)).

5.22.2.3. Burkitt's Lymphoma

Burkitt's lymphoma is an aggressive B cell lymphoma typically observedin children and young adults and is usually associated with bulkydisease of the jaw and/or abdomen. Approximately 20% of patients havebone marrow involvement. An endemic form of Burkitt's lymphoma involvesEpstein-Barr virus (EBV) infection of malignant cells; the sporadic formis independent of EBV infection. A translocation of c-myc toimmunoglobulin loci, which results in deregulation of the c-myc gene, ischaracteristic of this disease (t(8;14)(q24;q32)). Interestingly,deletions of the c-myc sequences appear to be involved in the sporadicform of the disease, while the endemic form usually involves pointmutations or insertions. (See, V. Pappa, et al., Molecular Biology, pp.133-157, In Malignant Lymphoma, B. Hancock, et al., eds., OxfordUniversity Press, New York, N.Y. (2000)). Burkitt's lymphoma is alsocharacterized by SHM, and the malignant cells have a gene expressionprofile similar to GC B cells, suggesting that this malignancy isderived from GC B cells.

Immunophenotype of Burkett's lymphoma shows the cells of this diseaseexpress CD19, CD20, CD22, and CD79a, but not CD5, CD23, cyclin D orterminal deoxynucleotidyl transferase. Frequently, these cells arepositive for CD10 and BCL6 and usually negative for BCL2. (See, I.Magrath, et al., Burkitt's Lymphoma, pp. 477-501, In Non-Hodgkin'sLymphomas, P. Mauch, et al., eds., Lippincott Williams & Wilkins,Philadelphia, Pa. (2004)).

High grade B cell Burkitt's-like lymphoma is a lymphoma borderlinebetween Burkitt's lymphoma and large B cell lymphoma. The cells of thislymphoma express CD19, CD20, and CD22 but expression of CD10, which isnearly always present in true Burkitt's lymphoma, is frequently absent.Because of this and other characteristics, some believe this lymphomashould be classified as a diffuse large B cell lymphoma. (See, K.Maclennan, Diffuse Aggressive B cell Lymphoma, pp. 49-54, In MalignantLymphoma, B. Hancock, et al., eds., Oxford University Press, New York,N.Y. (2000)).

Diagnosis of Burkitt's lymphoma generally relies on detection of thetranslocation associated with this lymphoma; thus, conventionalcytogenetic analysis is usually performed. Long distance polymerasechain reaction techniques and fluorescent in situ hybridization (FISH)have been used to detect Ig-myc junctions in the translocations andother genetic alterations associated with this disease. (See, R.Siebert, et al., Blood 91:984-990 (1998); T. Denyssevych, et al.,Leukemia, 16:276-283 (2002)).

5.22.2.4. Diffuse Large B-Cell Lymphoma (DLBCL)

DLBCL is the most common non-Hodgkin's lymphoma and can arise from smallB cell lymphoma, follicular lymphoma or marginal zone lymphoma.Typically, patients present with lymphadenopathy; however, a largepercent of patients present in extranodal sites as well, withgastrointestinal involvement being the most common. Bone marrowinvolvement is observed in about 15% of patients. (See, Armitage, etal., Diffuse Large B cell Lymphoma, pp. 427-453, In Non-Hodgkin'sLymphomas, P. Mauch, et al., eds., Lippincott Williams & Wilkins,Philadelphia, Pa. (2004)). Heterogeneity in clinical, biological andmorphological characteristics makes this group of lymphomas difficult tosubclassify. However, two distinct subgroups have been identified withone expressing genes characteristic of germinal center B cells(GC-DLBCL) and the other overexpressing genes in peripheral blood Bcells. Survival rates are significantly better for patients withGC-DLBCL than those with activated B cell type (ABC)-DLBCL. (See, W.Chan, Archives of Pathology and Laboratory Medicine 128(12): 1379-1384(2004)).

DLBCLs express the cell surface antigens CD19, CD20, CD22, and CD79a.CD10 is expressed in the large majority of cases and CD5 expression isobserved in about 10% of cases. (See, K. Maclennan, Diffuse Aggressive Bcell Lymphoma, pp. 49-54, In Malignant Lymphoma, B. Hancock, et al.,eds., Oxford University Press, New York, N.Y. (2000)). DLBCL is oftenmarked by abnormalities of BCL6 and/or translocations of BCL2 to the IgHlocus. GC B cell like (GC) DLBCL is characterized by SHM with highlymutated immunoglobulin genes and ongoing SHM in malignant clones with aGC B cell-like gene expression profile. Most GC DLBCL have undergoneimmunoglobulin class switching. ABC-DLBCL is characterized by high levelexpression of NF-KB target genes including BCL2, interferon regulatoryfactor 4, CD44, FLIP and cyclin D. SHM, but not ongoing SHM, is present,and ABC-DLBCL does not have a GC B cell gene expression profile. Almostall ABC-DLBCL express a high level of IgM.

5.22.2.5. Extranodal Marginal-Zone Lymphoma

Extranodal marginal-zone lymphoma is an extranodal lymphoma that occursin organs normally lacking organized lymphoid tissue (e.g., stomach,salivary glands, lungs and thyroid glands). It is largely a disease thataffects older adults with a median age of over 60 years. Often, chronicinflammation or autoimmune processes precede development of thelymphoma. Gastric mucosal-associated lymphoid tissue (MALT) lymphoma,the most common type of marginal-zone lymphoma, is associated withHelicobacter pylori infection. Studies have shown a resolution ofsymptoms with eradication of the H. pylori infection following anantibiotic regimen. The presenting symptoms for gastric MALT lymphomainclude nonspecific dyspepsia, epigastric pain, nausea, gastrointestinalbleeding and anemia. Systemic symptoms are uncommon, as are elevatedlevels of lactate acid dehydrogenase. (See, J. Yahalom, et al.,Extranodal Marginal Zone B cell Lymphoma of Mucosa-Associated LymphoidTissue, pp. 345-360, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds.,Lippincott Williams & Wilkins, Philadelphia, Pa. (2004); J. Radford,Other Low-Grade Non-Hodgkin's Lymphomas, pp. 325-330, In MalignantLymphoma, B. Hancock, et al., eds., Oxford University Press, New York,N.Y. (2000). Systemic B symptoms include fevers greater than 38° C. forlonger than 2 weeks without sign of infection, night sweats, extremefatigue or unintentional weight loss of greater than or equal to 10% ofbody weight over the previous 6 months).

The immunophenotype of MALT lymphoma is characterized by expression ofCD19, CD20, CD79a, CD21 and CD35 and lack of expression of CD5, CD23,and CD10. About half of MALT lymphomas express CD43. The immunoglobulintypically expressed in the tumor cells of this disease is IgM while IgDis not expressed. These features are critical in distinguishing thislymphoma from other small B cell lymphomas such as mantle cell lymphoma,lymphocytic lymphoma and follicular lymphoma. Trisomy 3 has beenreported in 60% of MALT lymphoma cases. In 25-40% of gastric andpulmonary MALT lymphomas a t(11;18) is observed. This translocation isobserved much less frequently in other MALT lymphomas. T(11;18) isassociated with nuclear expression of BCL10. (See, J. Yahalom, et al.,Extranodal Marginal Zone B cell Lymphoma of Mucosa-Associated LymphoidTissue, pp. 345-360, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds.,Lippincott Williams & Wilkins, Philadelphia, Pa. (2004)). Marginal-zonelymphomas are generally characterized by SHM and ongoing SHM.

Diagnostic procedures include immunophenotyping or flow cytometry todetermine the identity of the cell surface markers. In addition,molecular genetic analysis should be done to determine the presence oft(11;18) as this is an indicator that the disease will not respond toantibiotics. Histology can be used to determine the presence of H.pylori. Additional tests should include a complete blood count, basicbiochemical tests including that for lactate acid dehydrogenase; CTscans of the abdomen, chest and pelvis and a bone marrow biopsy. (See,J. Yahalom, et al., Extranodal Marginal Zone B cell Lymphoma ofMucosa-Associated Lymphoid Tissue, pp. 345-360, In Non-Hodgkin'sLymphomas, P. Mauch, et al., eds., Lippincott Williams & Wilkins,Philadelphia, Pa. (2004)).

5.22.2.6. Nodal Marginal Zone B Cell Lymphoma

Nodal Marginal Zone B cell Lymphoma is a relatively newly classifiedlymphoma thus little has been published on it. It is a primary nodal Bcell lymphoma sharing genetic and morphological characteristics withextranodal and splenic marginal zone lymphomas, but does not localize tothe spleen or extranodally. Hepatitis C virus has been reported to beassociated with this lymphoma as has Sjogren's syndrome. (See, F.Berger, et al., Nodal Marginal Zone B cell Lymphoma, pp. 361-365, InNon-Hodgkin's Lymphomas, P. Mauch, et al., eds., Lippincott Williams &Wilkins, Philadelphia, Pa. (2004)).

Nodal marginal zone lymphoma has a heterogeneous cytology andmorphology. Due to its relatively high proportion of large cells thislymphoma, unlike the other marginal lymphomas (splenic and extranodal),cannot be classified as true low grade B cell lymphoma. The genetic andimmunological phenotype of nodal marginal zone lymphoma includesexpression of CD19, CD20, CD22, BCL2, sIgM and cytoplasmic IgG (cIg).These cells do not express CDS, CD10, CD23, CD43 or cyclin D1. Thetranslocation characteristic of MALT lymphoma, t(11;18), is not observedfor nodal marginal zone lymphoma. These characteristics aid in thedifferential diagnosis of this lymphoma from other small B celllymphomas. (See, F. Berger, et al., Nodal Marginal Zone B cell Lymphoma,pp. 361-365, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds.,Lippincott Williams & Wilkins, Philadelphia, Pa. (2004)).

5.22.2.7. Splenic Marginal Zone Lymphoma

Splenic Marginal Zone Lymphoma is an indolent micro-nodular B celllymphoma with a characteristic clinical presentation of prominentsplenomegaly and infiltration of the peripheral blood and the bonemarrow. In addition, a relatively high level of liver involvement hasbeen reported. A role for hepatitis C virus has been postulated for thislymphoma. The immunophenotype of splenic marginal zone lymphoma istypically CD19⁺, CD20^(±), IgD⁺, BCL2⁺, p27⁺, CD3⁻, CD5⁻, CD10⁻, CD23⁻,CD38⁻, CD43⁻, BCL-6⁻, and cyclin D1⁻. Genetic characteristics include a7q deletion, p53 alterations and SHM. (See, M. Piris, et al., SplenicMarginal Zone Lymphoma, pp. 275-282, In Non-Hodgkin's Lymphomas, P.Mauch, et al., eds., Lippincott Williams & Wilkins, Philadelphia, Pa.(2004)).

Diagnosis generally relics on immunophenotyping to determine theidentity of the cell surface markers. Genetic and biochemical analysis,in combination with data on cell surface markers, help to differentiatethis lymphoma from other small B cell lymphomas. (See, M. Piris, et al.,Splenic Marginal Zone Lymphoma, pp. 275-282, In Non-Hodgkin's Lymphomas,P. Mauch, et al., eds., Lippincott Williams & Wilkins, Philadelphia, Pa.(2004)).

5.22.2.8. Acute (B Cell) Lymphocytic Leukemia (ALL)

ALL is a marrow-based neoplasm largely affecting children with thehighest incidence between 1-5 years. Most common symptoms atpresentation include fatigue, lethargy, fever and bone and joint pain.Fatigue and lethargy correlates with the degree of anemia present. Anelevated white blood cell count is common at presentment. Radiographs ofthe chest often show skeletal lesions. Extramedullary spread is commonand involves the central nervous system, testes, lymph nodes, liver,spleen and kidney. Anterior mediastinal masses are observed in onlyabout 5-10% of newly diagnosed cases. (See, J. Whitlock, et al., AcuteLymphocytic Leukemia, pp. 2241-2271, In Wintrobe's Clinical Hematology,Tenth Edition, G. Lee, et al., eds. Williams & Wilkins, Baltimore, Md.(1999)).

The immunophenotype of ALL is CD10⁺, CD19⁺, CD20⁺, CD22, and CD24⁺ Prc-Bcell ALL cells express cytoplasmic but not surface immunoglobulin, whilemature B cell ALL (which accounts for only 1-2% of ALL cases) isdistinguished from other leukemias of B cell lineage by the expressionof surface immunoglobulin. Cytogenetic characteristics of ALL includest(8;14), t(2;8) and t(8;22). Although rarely detected at the cytogeneticlevel t(12;21) may be the most common cytogenetic abnormality associatedwith childhood ALL (observed in about 25% of cases). (See, M. Kinney, etal., Classification and Differentiation of the Acute Leukemias, pp.2209-2240, In Wintrobe's Clinical Hematology, Tenth Edition, G. Lee, etal., eds. Williams & Wilkins, Baltimore, Md. (1999); J Whitlock, et al.,Acute Lymphocytic Leukemia, pp. 2241-2271; In Wintrobe's ClinicalHematology, Tenth Edition, G. Lee, et al., cds. Williams & Wilkins,Baltimore, Md., (1999)).

Precise diagnosis of acute leukemia usually relies on a bone aspirateand biopsy. Aspirate smears are used for morphological, immunologicaland cytological assessments. The demonstration of lymphoblasts in thebone marrow is diagnostic of ALL. The presence of greater than 5%leukemic lymphoblast cells in the bone marrow confirms ALL diagnosis butmost require greater than 25% for a definitive diagnosis. Lumbarpunctures are used to diagnose central nervous system involvement. Serumuric acids levels and serum lactate dehydrogenase levels have been foundto be elevated in ALL. (See, M. Kinney, et al., Classification andDifferentiation of the Acute Leukemias, pp. 2209-2240, In Wintrobe'sClinical Hematology, Tenth Edition, G. Lee, et al., eds. Williams &Wilkins, Baltimore, Md. (1999); J. Whitlock, et al., Acute LymphocyticLeukemia, pp. 2241-2271; In Wintrobe's Clinical Hematology, TenthEdition, G. Lee, et al., eds. Williams & Wilkins, Baltimore, Md.,(1999)).

5.22.2.9. Chronic Lymphocytic Leukemia (CLL)/Small B Cell LymphocyticLymphoma (SLL)

CLL/SLL is the most common type of leukemia. When the disease involvesthe peripheral blood and bone marrow it is referred to as CLL. However,when the lymph nodes and other tissues are infiltrated by cells that areimmunologically and morphologically identical to those in CLL, but whereleukemic characteristics of the disease are absent, then the disease isreferred to as SLL. This disease largely afflicts the elderly with agreater incidence of the disease occurring in men than women. Painlesslymphadenopathy is the most common finding at presentation.Hypogammaglobulinemia is common with most cases of CLL/SLL exhibitingreduced levels of all immunoglobulins rather than any particularsubclass of immunoglobulins. Asymptomatic patients are frequentlydiagnosed during routine blood counts (lymphocyte count of over5000×10⁹/L). As many as 20% of CLL/SLL cases report B symptoms. Anadditional diagnostic feature is infiltration of the bone marrow by morethan 30% by immature lymphocytes. Lymph node biopsies generally showinfiltration of involved nodes with well-differentiated lymphocytes.Autoimmune phenomena are often associated with CLL/SLL includingautoimmune hemolytic anemia and immune thrombocytopenia. (See, J.Gribben, et al., Small B cell Lymphocytic Lymphoma/Chronic LymphocyticLeukemia and Prolymphocytic Leukemia, pp. 243-261, In Non-Hodgkin'sLymphomas, P. Mauch, et al., eds., Lippincott Williams & Wilkins,Philadelphia, Pa. (2004); K. Maclennan, Diffuse Indolent B cellNeoplasms, pp. 43-47, In Malignant Lymphoma, B. Hancock, et al., eds.,Oxford University Press, New York, N.Y. (2000); Clinical Oncology, A.Neal, et al., Neal, Hoskin and Oxford University Press, co-publ., NewYork, N.Y. (2003)).

In contrast with many of the low-grade B cell malignancies, nonrandomreciprocal translocations are rarely found in CLL/SLL. However, othercytogenetic abnormalities have been reported including deletions at13q14, 11q22-23 and 17q13, with the latter two involving the p53 locus.Approximately 20% of cases exhibit trisomy 12. An elevated level of β-2microglobulin, higher levels of CD38 expression and the production oftumor necrosis factor-alpha are all characteristic of CLL/SLL. Theimmunophenotype of CLL/SLL is very diagnostic and includes weakexpression of surface immunoglobulin usually IgM, or IgM and IgG, aswell as expression of the cell antigens CD19, CD22, CD20 and usually CD5and CD23. (See, J. Gribben, et al., Small B cell LymphocyticLymphoma/Chronic Lymphocytic Leukemia and Prolymphocytic Leukemia, pp.243-261, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds., LippincottWilliams & Wilkins, Philadelphia, Pa. (2004); K. Maclennan, DiffuseIndolent B cell Neoplasms, pp. 43-47, In Malignant Lymphoma, B. Hancock,et al., eds., Oxford University Press, New York, N.Y. (2000)).

5.22.2.10. B Cell Prolymphocytic Leukemia (PLL)

PLL, once considered a variant of CLL, is now understood to be adistinct disease. PLL is generally a disease of elderly men and ischaracterized by a very high white blood cell count (greater than200×10⁹/L) and splenomegaly. Additional symptoms include anemia andthrombocytopenia. Prolymphocytes in PLL comprise more than 55% of thecells in the blood and bone marrow. In contrast with CLL, autoimmunephenomena are rarely observed in PLL. (See, J. Gribben, et al., Small Bcell Lymphocytic Lymphoma/Chronic Lymphocytic Leukemia andProlymphocytic Leukemia, pp. 243-261, In Non-Hodgkin's Lymphomas, P.Mauch, et al., eds., Lippincott Williams & Wilkins, Philadelphia, Pa.(2004)).

The immunophenotype of PLL is characterized by expression of CD19, CD21,CD22, CD24 and FMC7. The cells of PLL do not express CD23 and most donot express CD5. PLL cells exhibit complex chromosomal abnormalities,with deletions at 13q14 and 11q23 being some of the most frequent. Thepattern of p53 mutation in PLL cells is different from that observed forCLL. Differential diagnosis usually relies on complete blood count,histological, immunophenotypic, and genetic analyses. (See, J. Gribben,et al., Small B cell Lymphocytic Lymphoma/Chronic Lymphocytic Leukemiaand Prolymphocytic Leukemia, pp. 243-261, In Non-Hodgkin's Lymphomas, P.Mauch, et al., eds., Lippincott Williams & Wilkins, Philadelphia, Pa.(2004)).

5.22.2.11. Hairy Cell Leukemia (HCL)

HCL is a rare, indolent chronic leukemia affecting more men than womenand largely those of middle age. The typical symptoms include massivesplenomegaly and pancytopenia. The peripheral blood and bone marrowcontain the typical “hairy cells,” which are B lymphocytes withcytoplasmic projections. Over 90% of HCL patients have bone marrowinfiltration. (See, Clinical Oncology, A. Neal, et al., Neal, Hoskin andOxford University Press, co-publ., New York, N.Y. (2003); J. Johnston,Hairy Cell Leukemia, pp. 2428-2446, In Wintrobe's Clinical Hematology,Tenth Edition, G. Lee et al., eds. Williams & Wilkins, Baltimore, Md.(1999)).

Cytogenetic analysis has shown that clonal abnormalities are present in19% of cases and involve numerical and structural abnormalities ofchromosomes 5, 7 and 14. The serum level of TNF-α is elevated in hairycell leukemia and correlates with tumor burden. Hairy cell leukemiacells express surface immunoglobulins (1gG and 1gM) and CD11 c, CD19,CD20, CD22 and typically CD25. In addition, FMC7, HC-2 and CD103 areexpressed. HCL cells do not express CD5 or CD10. Diagnosis generallyinvolves the use of bone marrow aspirates, cytogenetics, blood smearsand immunophenotyping. (See, Clinical Oncology, A. Neal, et al., Neal,Hoskin and Oxford University Press, co-publ., New York, N.Y. (2003); J.Johnston, Hairy Cell Leukemia, pp. 2428-2446, In Wintrobe's ClinicalHematology, Tenth Edition, G. Lee et al., eds. Williams & Wilkins,Baltimore, Md. (1999)).

5.22.2.12. Precursor B Cell Lymphoblastic Lymphoma/Pre-B Cell AcuteLymphoblastic Leukemia/Lymphoblastic Lymphoma

Precursor B cell lymphoblastic lymphoma/pre-B cell acute lymphoblasticleukemia/Lymphoblastic lymphoma is a disease of precursor T or B cells.The T and B cell lymphoblastic lymphomas are morphologically identical,but clinical distinctions may be made based on degree of bone marrowinfiltration or bone marrow involvement. 85-90% of lymphoblasticlymphomas are T-cell derived with the remainder being B cell derived.Lymphoblastic lymphoma has a median age of 20 years with a malepredominance. Peripheral lymph node involvement is a common feature atpresentation, occurring especially in the cervical, supraclavicular andaxillary regions. This disease frequently presents with bone marrowinvolvement. Central nervous system is less common at presentment butoften appears in cases of relapse. Other sites of involvement caninclude liver, spleen, bone, skin, pharynx and testes (See, J.Sweetenham, et al., Precursor B-and T-Cell Lymphoblastic Lymphoma, pp.503-513, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds., LippincottWilliams & Wilkins, Philadelphia, Pa. (2004)).

Precursor B cell lymphoblastic lymphomas express immature markers B cellmarkers such as CD99, CD34 and terminal deoxynucleotidyl transferase.These cells also express CD79a, CD19, CD22 and sometimes CD20 andtypically lack expression of CD45 and surface immunoglobulin.Translocations at 11q23, as well as t(9;22)(q34;q11.2) andt(12;21)(p13;q22), have been associated with poor prognosis. Goodprognosis is associated with hyperdiploid karyotype, especially thatassociated with trisomy 4, 10, and 17 and t(12;21)(p13;q22). (See, J.Sweetenham, et al., Precursor B-and T-Cell Lymphoblastic Lymphoma, pp.503-513, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds., LippincottWilliams & Wilkins, Philadelphia, Pa. (2004)).

Diagnostic tests include lymph node biopsies, blood tests, x-rays, CTscans, and lumbar punctures to examine the cerebralspinal fluid formalignant cells.

5.22.2.13. Primary Mediastinal Large B Cell Lymphoma

Primary mediastinal large B cell lymphoma is a diffuse large B celllymphoma occurring predominantly in young women and characterized by alocally invasive anterior mediastinal mass originating in the thymus.Distant spread to peripheral nodes and bone marrow involvement isunusual. Systemic symptoms are common. While this disease resemblesnodal large cell lymphomas, it has distinct genetic, immunological, andmorphological characteristics.

The immunophenotype of tumor cells of primary mediastinal large B celllymphoma are often surface immunoglobulin negative but do express such Bcell associated antigens as CD19, CD20, CD22, and CD79a. CD10 and BCL6are also commonly expressed. Expression of plasma cell associatedmarkers CD15, CD30, epithelial membrane antigen (EMA) is rare. BCL6 andc-nzyc gene arrangements are also uncommon. The presence of clonalimmunoglobulin rearrangements, immunoglobulin variable region and genehypermutation along with BCL6 hypermutation suggest that this lymphomaderives from a mature germinal center or post-germinal center B cell.The chromosomal translocations that seem to be associated with tumors ofthis disease are similar to those observed in other forms of diffuselarge cell lymphoma. (See, P. Zinzani, et al., Primary Mediastinal LargeB cell Lymphoma, pp. 455-460, In Non-Hodgkin's Lymphomas, P. Mauch, etal., eds., Lippincott Williams & Wilkins, Philadelphia, Pa. (2004)).

The diagnostic evaluation for primary mediastinal large B cell lymphomagenerally includes a complete physical examination, completehematological and biochemical analysis, total-body computerizedtomography and bone marrow biopsy. Gallium-67 scanning is a useful testfor staging, response to treatment and for assessment of relapse. (See,P. Zinzani et al., Primary Mediastinal Large B cell Lymphoma, pp.455-460, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds., LippincottWilliams & Wilkins, Philadelphia, Pa. (2004)).

5.22.2.14. Lymphoplasmacytic Lymphoma (LPL)/LymphoplasmacyticImmunocytoma/Waldstrom's Macroglobulinemia

LPL/Lymphoplasmacytic immunocytoma/Waldstrom's Macroglobulinemia is anodal lymphoma that is usually indolent, and often involves bone marrow,lymph nodes and spleen. This is generally a disease of older adults withmales slightly predominating. Most patients have monoclonal IgMparaprotein in their serum (>3 g/dL) resulting in hyperviscosity of theserum. Tumor cells have a plasmacytic morphology. A subset of LPL ischaracterized by recurrent translocations between chromosomes 9 and 14,which involves the PAX5 and immunoglobulin heavy-chain loci. LPL ischaracterized by SHM as well as ongoing SHM, and is believed to bederived from post-GC B cells. (See, A. Rohatiner, et al.,Lymphoplasmacytic Lymphoma and Waldstrom's Macroglobulinemia, pp.263-273, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds., LippincottWilliams & Wilkins, Philadelphia, Pa. (2004); K. Maclennan, DiffuseIndolent B cell Neoplasms, pp. 43-47, In Malignant Lymphoma, B. Hancock,et al., eds., Oxford University Press, New York, N.Y. (2000); A. Lal, etal., Role of Fine Needle Aspiration in Lymphoma, pp. 181-220, In W.Finn, et al., eds., Hematopathology in Oncology, Kluwer AcademicPublishers, Norwell, Mass. (2004)).

The immunophenotype of this disease shows expression of the B cellassociated antigens CD19, CD20, CD22, and CD79a and a lack of expressionof CD5, CD10, and CD23. Presence of strong surface immunoglobulin andCD20, the lack of expression of CD5, and CD23 and the presence ofcytoplasmic immunoglobulin are characteristics that aid indistinguishing this disease from chronic lymphocytic leukemia. Alsodiagnostic of this disease is t(9;14)(p13;q32). (See, A. Rohatiner, etal., Lymphoplasmacytic Lymphoma and Waldstrom's Macroglobulinemia, pp.263-273, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds., LippincottWilliams & Wilkins, Philadelphia, Pa. (2004); K. Maclennan, DiffuseIndolent B cell Neoplasms, pp. 43-47, In Malignant Lymphoma, B. Hancock,et al., eds., Oxford University Press, New York, N.Y. (2000); R.Chaganti, et al., Cytogenetics of Lymphoma, pp. 809-824, InNon-Hodgkin's Lymphomas, P. Mauch, et al., eds., Lippincott Williams &Wilkins, Philadelphia, Pa. (2004)).

Diagnostic tests typically include a complete blood count, renal andliver function tests, CT scans, biopsy and aspiration of the bonemarrow, protein electrophoresis to quantify and characterize theparaprotein and serum viscosity. Measurement of 132-microglobulin isused as a prognostic test. (See, A. Rohatiner, et al., LymphoplasmacyticLymphoma and Waldstrom's Macroglobulinemia, pp. 263-273, InNon-Hodgkin's Lymphomas, P. Mauch, et al., eds., Lippincott Williams &Wilkins, Philadelphia, Pa. (2004)).

5.22.2.15. Null-Acute Lymphoblastic Leukemia

Null-acute lymphoblastic leukemia is a subset of ALL which lacks B- orT-cell characteristics. Phenotypic analysis of leukemic blasts shows atypical null ALL pattern, i.e., CD10 (common ALL antigen)-negative,strongly HLA-DR-positive, and CD19 (B4)-positive (see Katz et al. (1988)Blood 71(5):1438-47).

5.22.2.16. Hodgkin's Lymphoma

Hodgkin's lymphoma usually arises in the lymph nodes of young adults. Itcan be divided into classical subtype and a less common nodularlymphocytic predominant subtype. The classical type exhibits SHM, butnot ongoing SHM, and does not have a GC B cell gene expression profile.The nodular lymphocyte predominant type, in contrast, is characterizedby SHM and ongoing SHM and a GC B cell gene expression profile. Whilethe two types differ clinically and biologically, they do share certainfeatures such as a lack of neoplastic cells within a background ofbenign inflammatory cells. B. Schnitzer et al., Hodgkin Lymphoma, pp.259-290, In W. Finn and L. Peterson, eds., Hematopathology in Oncology,Kluwer Academic Publishers, Norwell, Mass. (2004)).

The most common features at presentation are painless enlargement oflymph nodes, usually in the neck, but occasionally in the inguinalregion. Waxing and waning of nodes is also characteristic of thisdisease. B symptoms are observed in about one-third of patients.Isolated extranodal involvement is rare and in cases where disseminationhas occurred extranodal involvement is observed about 10-20% of thetime. (See, P. Johnson et al., Hodgkin's Disease: Clinical Features, pp.181-204, In Malignant Lymphoma, B. Hancock, et al., eds., OxfordUniversity Press, New York, N.Y. (2000)).

Reed-Sternberg (RS) cells are the malignant cells of Hodgkin's lymphoma.RS cells and their variants express CD15, CD25, CD30 and transferrinreceptor. In addition these cells express polyclonal cytoplasmicimmunoglobulin. In most cases of Hodgkin's lymphoma the RS cells do notexpress CD45, a feature that aids in distinguishing this disease fromnon-Hodgkin's Lymphomas. Epstein Barr virus has been demonstrated to bepresent in Reed-Sternberg cells in about one-half of Hodgkin's lymphomacases but its role is unclear.

Diagnosis is most frequently made by lymph node biopsy. Additionaldiagnostic tests include a full blood count (often hematological testsare normal; white blood cell counts of less than 1.0×109/L are seen inabout 20% of cases), erythrocyte sedimentation rate (often elevated inadvanced stages of the disease), biochemical tests includingelectrolytes, urea, creatinine, urate, calcium (hypercalcemia is rarebut when present is associated with extensive bone involvement), liverblood tests, lactate dehydrogenase (elevated levels often associatedwith advanced disease), albumin and beta2-microglobulin (I32-M).Lymphanigiograms and chest x-rays and CT scans of the chest, abdomen andpelvis are important in identifying abnormal lymph nodes and the extentof extranodal involvement. Bone marrow biopsies are typically consideredoptional as bone marrow involvement is unusual and the results of suchbiopsies appear not to affect clinical management or prognosis.Splenechtomies are not usually performed today as it rarely influencesmanagement and CT or MRI imaging provides information on splenic status.Significantly elevated levels of p55, TNF and sICAM-1 are correlated tothe stage of the disease, presence of symptoms and complete responserate. (See, P. Johnson, et al., Hodgkin's Disease: Clinical Features,pp. 181-204, In Malignant Lymphoma, B. Hancock, et al., eds., OxfordUniversity Press, New York, N.Y. (2000); Clinical Oncology, A. Neal, etal., Neal, Hoskin and Oxford University Press, co-publ., New York, N.Y.(2003); R. Stein, Hodgkin's Disease, pp. 2538-2571, In Wintrobe'sClinical Hematology, Tenth Edition, G. Lee et al., eds. Williams &Wilkins, Baltimore, Md. (1999)).

5.22.2.17. Multiple Myeloma

Multiple myeloma is a malignancy of plasma cells. Neoplastic cells arelocated in the bone marrow, and osteolytic bone lesions arecharacteristic. Reciprocal chromosomal translocations between one of theimmunoglobulin loci and a variety of other genes, e.g., cyclin D1,cyclin D3, c-MAF, MMSET (multiple myeloma SET-domain protein) orfibroblast growth factor receptor 3 are believed to be the primaryoncogenic events. Multiple myeloma is characterized by SHM, and theputative cell of origin is a post-GC B cell. Multiple myeloma istypically first identified by symptoms such as recurrent infection,fatigue, pain, and kidney problems and is confirmed with clinicaltesting (see, for example, Cancer: Principles and Practice of Oncology.6th edition. DeVita, V. T., Hellman, S. and Rosenberg, S. A. editors.2001 Lippincott Williams and Wilkins Philadelphia, Pa. 19106 pp.2465-2499).

In certain embodiments, patients who are candidates for treatment bycompositions and methods of the invention can undergo further diagnostictests on blood and/or urine to confirm the diagnosis or suspicion ofmultiple myeloma including, but not limited to, complete blood count(CBC) tests to determine if the types of cells reported in a CBC arewithin their normal ranges which are well known in the art, bloodchemistry profile to determine whether levels of various bloodcomponents, such as albumin, blood urea nitrogen (BUN), calcium,creatinine, and lactate dehydrogenase (LDH), deviate from standardvalues. Serum levels of beta2-microglobulin (B2-M) can also be examinedand surrogate markers for IL-6, a growth factor for myeloma cells.Urinalysis can be used to measure the levels of protein in the urine.Electrophoresis can be used to measure the levels of various proteins,including M protein in the blood (called serum protein electrophoresis,or SPEP) or urine (called urine electrophoresis, or UEP). An additionaltest, called immunofixation electrophoresis (IFE) orimmunoelectrophoresis, may also be performed to provide more specificinformation about the type of abnormal antibody proteins present.Assessing changes and proportions of various proteins, particularly Mprotein, can be used to track the progression of myeloma disease andresponse to treatment regimens. Multiple myeloma is characterized by alarge increase in M protein which is secreted by the myeloma tumorcells.

Diagnostic tests on bone can also be conducted to confirm the diagnosisor suspicion of multiple myeloma including, but not limited to, X-raysand other imaging tests—including a bone (skeletal) survey, magneticresonance imaging (MRI), and computerized axial tomography (CAT), alsoknown as computed tomography (CT)—can assess changes in the bonestructure and determine the number and size of tumors in the bone. Bonemarrow aspiration or bone marrow biopsy can be used to detect anincrease in the number of plasma cells in the bone marrow. Aspirationrequires a sample of liquid bone marrow, and biopsy requires a sample ofsolid bone tissue. In both tests, samples can be taken from the pelvis(hip bone). The sternum (breast bone) can also be used for aspiration ofbone marrow.

Patients with multiple myeloma are typically categorized into thefollowing three groups that help define effective treatment regimens.Monoclonal gammopathy of undetermined significance (MGUS) is typicallycharacterized by a serum M protein level of less than 3 g/dL, bonemarrow clonal plasma cells of less than 10%, no evidence of other B celldisorders, and no related organ or tissue impairment, such ashypercalccmia (increased serum calcium levels), impaired kidney functionnoted by increased serum creatinine, anemia, or bone lesions.Asymptomatic myelomas are typically stage I and includes smolderingmultiple myeloma (SMM) and indolent multiple myeloma (IMM). SMM ischaracterized by serum M protein greater than or equal to 3 g/dL and IMMis characterized by bone marrow clonal plasma cells greater than orequal to 10% of the bone marrow cells. Symptomatic myeloma ischaracterized by M protein in serum and/or urine and includes Stage 11multiple myeloma characterized by the presence of bone marrow clonalplasma cells or plasm acytoma and Stage III multiple myelomacharacterized by related organ or tissue impairment.

Osteosclerotic myeloma is a component of the rare POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy andskin lesions). Peak incidence is at 40 to 50 years of age. Systemicfeatures include skeletal lesions, marrow-plasma cells <5%, a normalCBC, increased platelets, and organomegaly. The CSF has a high proteinwith no cells present. The M-protein levels are low (<3 g/dl, median=1.1g/dl); heavy chain class—usually α or γ; light chain class—usually λ;rare urine monoclonal and occasional cryoglobulinemia. Neuropathy occursin 50% of the patients with weakness both proximal and distal, sensoryloss is greater in larger than small fibers; and demyelination and longdistal latency.

Smoldering multiple myeloma patients generally present with stabledisease for months/years; no anemia, bone lesions, renal insufficiencyor hypercalcemia; have >10% plasma cells in bone marrow and monoclonalserum protein. The criteria for smoldering multiple myeloma iscompatible with the diagnosis of multiple myeloma; however, there is noevidence of progressive course. These are cases with a slow progression,the tumor cell mass is low at diagnosis and the percentage of bonemarrow plasma cells in S phase is low (<0.5%). Characteristic clinicalfeatures include: serum M protein levels >3 g/dL and/or bone marrowplasma cells ≥10%; absence of anemia, renal failure, hypercalcemia,lytic bone lesions.

Indolent (or asymptomatic) multiple myeloma is a multiple myelomadiagnosed by chance in the absence of symptoms, usually after screeninglaboratory studies. Indolent multiple myeloma is similar to smolderingmyeloma but with few bone lesions and mild anemia. Most cases ofindolent multiple myeloma develop overt multiple myeloma within 3 years.Diagnostic criteria are the same as for multiple myeloma except: no bonelesions or one asymptomatic lytic lesion (X-ray survey); M componentlevel <3 g/dL for IgG, 2 g/dL for IgA urine light chain <4 g/24 h;hemoglobin >10 g/dl, serum calcium normal, serum creatinine <2 mg/dL,and no infections.

5.22.2.18. Solitary Plasmacytoma

Solitary plasmacytoma is one of a spectrum of plasma cell neoplasmswhich range from benign monoclonal gammopathy to solitary plasmacytomato multiple myeloma. Approximately seventy percent of all solitaryplasmacytoma cases eventually result in multiple myeloma. These diseasesare characterized by a proliferation of B cells which produce thecharacteristic paraprotein. Solitary plasmacytoma results in aproliferation of clonal plasma cells in a solitary site, usually asingle bone or extramedullary tissue site. Diagnostic criteria ofsolitary plasmacytoma include a histologically confirmed single lesion,normal bone biopsy, negative skeletal survey, no anemia, normal calciumand renal function. Most cases exhibit minimally elevated serumM-protein (paraprotein). The median age at diagnosis is 50-55, about5-10 years younger than the median age for multiple myeloma. (See, C.Wilson, The Plasma Cell Dycrasias, pp. 113-144, In W. Finn and L.Peterson, eds., Hematopathology in Oncology, Kluwer Academic Publishers,Norwell, Mass. (2004), S. Chaganti, et al., Cytogenetics of Lymphoma,pp. 809-824, In Non-Hodgkin's Lymphomas, P. Mauch, et al., eds.,Lippincott Williams & Wilkins, Philadelphia, Pa., (2004)).

The immunophenotypic and genetic features of plasmacytoma appear to besimilar to multiple myeloma.

5.22.2.19. Light Chain Disease/Light Chain Deposition Disease (LCDD)

LCDD is a plasma cell dycrasias disorder caused by the over-synthesis ofimmunoglobulin light chains (usually kappa light chains) that aredeposited in tissues. Patients commonly present with organ dysfunction,weakness, fatigue and weight loss. In approximately 80% of cases of LCDDa monoclonal immunoglobulin is detected. Detection of monoclonal kappalight chains using immunofluorescent techniques is limited by thetendency of light chains to give excess background staining, therefore,ultrastructural immunogold labeling may be necessary. (See, C. Wilson,The Plasma Cell Dycrasias, pp. 113-144, In W. Finn and L. Peterson,cds., Hematopathology in Oncology, Kluwer Academic Publishers, Norwell,Mass. (2004)).

5.22.2.20. Plasma Cell Leukemia (PCL),

PCL, a plasma cell dycrasias, is a rare aggressive variant of multiplemyeloma. The criteria for plasma cell leukemia is a peripheral bloodabsolute plasma cell count of greater than 2×109/L or plasma cellsgreater than 20% of white blood cells. Determination of the presence ofa CD138+ population with cytoplasmic light chain restriction by flowcytometry will distinguish PCL from lymphoid neoplasm with plasmacyticfeatures. PCL cells are also characterized by the lack of surface lightchain, CD19 and CD22 expression, and either no or weak expression ofCD45. About 50% of cases of PCL express CD20 and about 50% lackexpression of CD56. The genetic abnormalities observed in PCL patientsare the same as those observed for multiple myeloma patients but theyare found at higher frequency in PCL. (See, C. Wilson, The Plasma CellDycrasias, pp. 113-144, In W. Finn and L. Peterson, eds.,Hematopathology in Oncology, Kluwer Academic Publishers, Norwell, Mass.,(2004)).

Plasma cell leukemia has two forms: if initial diagnosis is based onleukemic phase of myeloma then the primary form is present, otherwise itis secondary. Primary plasma cell leukemia is associated with a youngerage, hepatosplenomegaly, lymphadenopathy, and fewer lytic bone lesionsbut poorer prognosis than the secondary form. The peripheral blood ofplasma cell leukemic patients has greater than 20% plasma cells withabsolute count of 2000/ml or more.

5.22.2.21. Monoclonal Gammopathy of Unknown Significance (MGUS)

MGUS is a relatively common condition characterized by the presence ofelectrophoretically homogeneous immunoglobulins or benign M-components.The occurrence of this condition appears to increase with age. Mostindividuals carrying the M-components never develop malignant plasmacell dycrasias, such as multiple myeloma. However, some individuals withthis condition have associated malignant conditions. When symptomatic,patients can have enlarged liver or spleen and pleuroneuropathy. (See,J. Foerster, Plasma Cell Dycrasias: General Considerations, pp.2612-2630, In Wintrobe's Clinical Hematology, Tenth Edition, G. Lee etal., eds. Williams & Wilkins, Baltimore, Md. (1999)).

MGUS can be differentiated from multiple myeloma by the presence ofincreased number of monoclonal plasma cells circulating in theperipheral blood. The serological characteristics of M-components areidentical to other plasma cell dycrasias conditions, however, the totalconcentration of M-component is usually less than 30 g/L. Theparaprotein is usually IgG; however multiple paraproteins may be presentincluding IgG, IgA, IgM. The relative amount of each of the individualimmunoglobulin classes is typically proportional to that found in normalserum. Proteinemia or proteinuria is rare. Serial measurements ofM-protein levels in the blood and urine, and continued monitoring of theclinical and laboratory features (including protein electrophoresis) isthe most reliable method of differentiating MGUS from early stage plasmacell dycrasias. In Wintrobe's Clinical Hematology, Tenth Edition, G. Leeet al., eds. Williams & Wilkins, Baltimore, Md. (1999)).

5.22.2.22. Mature B Cell Malignancies:

In a further embodiment the invention can be practiced to treat mature Bcell malignancies including but not limited to follicular lymphoma,mantle-cell lymphoma, Burkitt's lymphoma, multiple myeloma, diffuselarge B-cell lymphoma (DLBCL) including germinal center B cell-like(GCB) DLBCL, activated B cell-like (ABC) DLBCL, and type 3 DLBCL,Hodgkin's lymphoma including classical and nodular lymphocytepre-dominant type, lymphoplasmacytic lymphoma (LPL), marginal-zonelymphoma including gastric mucosal-associated lymphoid tissue (MALT)lymphoma, and chronic lymphocytic leukemia (CLL) includingimmunoglobulin-mutated CLL and immunoglobulin-unmutatcd CLL.

5.22.2.23. Pre-B Cell Malignancies:

Further, CD19 is expressed earlier in B cell development than, forexample, CD20, and is therefore particularly suited for treating pre-Bcell and immature B cell malignancies, e g., in the bone marrow.Representative pre-B cell and immature B cell malignancies include butare not limited to mantle cell lymphoma, pre-B cell acute lymphoblasticleukemia, precursor B cell lymphoblastic lymphoma, and othermalignancies characterized by CD19 expression.

5.23. Patient Diagnosis and Therapeutic Regimens Transplantation

According to certain aspects of the invention, the treatment regimen anddose used with compositions and methods of the invention is chosen basedon a number of factors including, for example, clinical manifestationthat place a patient at risk for developing a humoral rejection, orclinical evidence that such a rejection is developing. The terms“humoral” and “antibody-mediated” are used interchangeably herein.

The criteria for assessing the risk that a patient will develop ahumoral rejection are established according to the knowledge and skillin the art. In one embodiment, a positive complement dependentcytotoxicity or antiglobulin enhanced complement dependent cytotoxicitycrossmatch indicates that a patient is at high risk for humoralrejection. In one embodiment, a positive crossmatch or a prior positivecomplement dependent cytotoxicity or anti-globulin enhanced complementdependent cytotoxicity crossmatch indicates that a patient is at anintermediate risk for humoral rejection. In one embodiment, a negativecrossmatch indicates that a patient is at a low risk for humoralrejection.

In another embodiment, a transplant recipient in need of prophylaxisagainst graft rejection may be identified as a patient or patientpopulation having detectable circulating anti-HLA alloantibodies priorto transplantation. In another example, the patient or patientpopulation is identified as having panel reactive antibodies prior totransplantation. The presence of detectable circulating anti-HLAalloantibodies in a transplant recipient post-transplantation can alsobe used to identify the patient or patient population in need oftreatment for humoral rejection according to the invention. The patientor patient population in need of treatment for humoral rejection canalso be identified according to other clinical criteria that indicatethat a transplant recipient is at risk for developing a humoralrejection or has already developed a humoral rejection. For example, atransplant recipient in need of treatment of humoral rejection may beidentified as a patient or population in an early stage of humoralrejection, such as a latent humoral response characterized bycirculating anti-donor alloantibodics. An early stage of humoralrejection may also be a silent reaction characterized by circulatinganti-donor alloantibodies and C4d deposition, or a subclinical rejectioncharacterized by circulating anti-donor alloantibodies, C4d deposition,and tissue pathology. In later stages, the recipient is identified as apatient or patient population presenting with clinical indications ofhumoral rejection characterized according to the knowledge and skill inthe art, for example, by circulating anti-donor alloantibodies, C4ddeposition, tissue pathology, and graft dysfunction.

The present invention provides compositions, therapeutic formulations,methods and regimens effective to reduce the incidence, severity, orduration of GVHD, a rejection episode, or post-transplantlymphoprolifcrativc disorder. In certain embodiments, compositions andmethods of the invention are effective to attenuate the host response toischemic reperfusion injury of a solid tissue or organ graft. In oneembodiment, compositions and methods of the invention are effective toprolong survival of a graft in a transplant recipient.

The present invention encompasses grafts that are autologous,allogeneic, or xenogeneic to the recipient. The types of graftsencompassed by the invention include tissue and organ grafts, includingbut not limited to, bone marrow grafts, peripheral stem cell grafts,skin grafts, arterial and venous grafts, pancreatic islet cell grafts,and transplants of the kidney, liver, pancreas, thyroid, and heart. Theterms “graft” and “transplant” are used interchangeably herein. In oneembodiment, the autologous graft is a bone marrow graft, an arterialgraft, a venous graft or a skin graft. In one embodiment, the allograftis a bone marrow graft, a corneal graft, a kidney transplant, apancreatic islet cell transplant, or a combined transplant of a kidneyand pancreas. In one embodiment, the graft is a xenograft, wherein thepossible animal donors include, but are not limited to pigs. Thecompositions and methods of the present invention may also be used tosuppress a deleterious immune response to a non-biological graft orimplant, including but not limited to an artificial joint, a stent, or apacemaker device.

Anti-CD19 antibodies, compositions, and methods of the invention may beused to treat or prevent GVHD, humoral rejection, or post-transplantlymphoproliferative disorder without regard to the particularindications initially giving rise to the need for the transplant or theparticular type of tissue transplanted.

Therapeutic formulations and regimens of the present invention aredescribed for treating human subjects diagnosed with autoimmunc diseasesor disorders, including but not limited to, rheumatoid arthritis, SLE,1TP, pemphigus-related disorders, diabetes, and scleroderma.

Appropriate treatment regimens can be determined by one of skill in theart for the particular patient or patient population. In particularembodiments, the treatment regimen is a pre-transplant conditioningregimen, a post-transplant maintenance regimen, or post-transplanttreatment regimen for an acute or a chronic rejection. In certainembodiments, the particular regimen is varied for a patient who isassessed as being at a high or intermediate risk of developing a humoralresponse, compared with the regimen for a patient who is assessed asbeing at a low risk of developing a humoral response.

In certain embodiments, the particular regimen is varied according tothe stage of humoral rejection, with more aggressive therapy beingindicated for patients at later stages of rejection. The stages ofhumoral rejection may be classified according to the knowledge and skillin the art. For example, the stages of humoral rejection may beclassified as one of stages I to IV according to the following criteria:Stage I Latent Response, characterized by circulating anti-donoralloantibodies, especially anti-HLA antibodies; Stage 11 SilentReaction, characterized by circulating anti-donor alloantibodies,especially anti-HLA antibodies, and C4d deposition, but withouthistologic changes or graft dysfunction; Stage III SubclinicalRejection: characterized by circulating anti-donor alloantibodies,especially anti-HLA antibodies, C4d deposition, and tissue pathology,but without graft dysfunction; Stage IV Humoral Rejection: characterizedby circulating anti-donor alloantibodies, especially anti-HLAantibodies, C4d deposition, tissue pathology, and graft dysfunction.

Dose response curves can be generated using standard protocols in theart in order to determine the effective amount of compositions of theinvention for use in a particular regimen, for example, in conditioningregimens prior to transplantation, and in post-transplantation regimensfor prophylaxis and treatment of GVHD, humoral rejection, orpost-transplantation lymphoproliferative disorders. In general, patientsat high risk for developing a humoral rejection and those alreadyexhibiting one or more clinical indicators of rejection will requirehigher doses and/or more frequent doses which may be administered overlonger periods of time in comparison to patients who are not at highrisk or who do not exhibit any indications of active rejection.

Anti-CD19 antibodies, compositions and methods of the invention may bepracticed to treat or prevent GVHD, humoral rejection, orpost-transplantation lymphoproliferative disorders, either alone or incombination with other therapeutic agents or treatment regimens. Othertherapeutic regimens for the treatment or prevention of GVHD, humoralrejection, or post-transplantation lymphoproliferative disorders maycomprise, for example, one or more of anti-lymphocyte therapy, steroidtherapy, antibody depletion therapy, immunosuppression therapy, andplasmapheresis.

Anti-lymphocyte therapy may comprise the administration to thetransplant recipient of anti-thymocyte globulins, also referred to asthymoglobulin. Anti-lymphocyte therapy may also comprise theadministration of one or more monoclonal antibodies directed against Tcell surface antigens. Examples of such antibodies include, withoutlimitation, OKT3™ (muromonab-CD3), CAMPATH™-1H (alemtuzumab),CAMPATH™-1G, CAMPATH™-1M, SIMULECT™ (basiliximab), and ZENAPAX™(daclizumab). In a specific embodiment, the anti-lymphocyte therapycomprises one or more additional antibodies directed against B cells,including, without limitation, RITUXAN™ (rituximab).

Steroid therapy may comprise administration to the transplant recipientof one or more steroids selected from the group consisting of cortisol,prednisone, methyl prednisolone, dexamethazone, and in domethacin. Oneor more of the steroids may be corticosteroids, including withoutlimitation, cortisol, prednisone, and methylprednisolone.

Antibody depletion therapy may include, for example, administration tothe transplant recipient of intravenous immunoglobulin. Antibodydepletion therapy may also comprise immunoadsorption therapy applied tothe graft ex vivo, prior to transplantation. Immunoadsorption may beaccomplished using any suitable technique, for example, protein Aaffinity, or antibody based affinity techniques using antibodiesdirected against T cell or B cell surface markers such as anti-CD3antibodies, anti-CD19 antibodies, anti-CD20 antibodies, and anti-CD19antibodies.

Immunosuppression therapy may comprise the administration of one or moreimmunosuppressive agents such as inhibitors of cytokine transcription(e.g., cyclosporin A, tacrolimus), nucleotide synthesis (e.g.,azathiopurine, mycophenolate mofetil), growth factor signal transduction(e.g., sirolimus, rapamycin), and the T cell interleukin 2 receptor(e.g., daclizumab, basiliximab). In a particular embodiment, animmunosuppressant agent used in combination with compositions andmethods of the invention includes one or more of the following:adriamycin, azathiopurine, busulfan, cyclophosphamide, cyclosporin A(“CyA”), cytoxin, fludarabinc, 5-fluorouracil, methotrexatc,mycophcnolatc mofctil (MOFETIL), nonsteroidal anti-inflammatories(NSAIDs), rapamycin, and tacrolimus (FK506).

Immunosuppressive agents may also comprise inhibitors of complement, forexample, soluble complement receptor-1, anti-05 antibody, or a smallmolecule inhibitor of Cls, for example as described in Buerke et al. (J.Immunol., 167:5375-80 (2001).

In one embodiment, compositions and methods of the invention are used incombination with one or more therapeutic regimens for suppressinghumoral rejection, including, without limitation, tacrolimus andmycophenolate mofetil therapy, immunoadsorption, intravenousimmunoglobulin therapy, and plasmapheresis.

5.23.1. Diagnosis and Clinical Criteria

The present invention provides antibodies, compositions and methods fortreating and preventing GVHD, humoral rejection, and post-transplantlymphoproliferative disorder in human transplant recipients.Compositions and methods of the invention can be used regardless of theparticular indications which gave rise to the need for a transplant.Similarly, the use of compositions and methods of the invention for thetreatment and prevention of GVHD, humoral rejection, and post-transplantlymphoproliferative disorders is not limited by the particular type oftissue which is intended for transplantation or which has beentransplanted.

In one embodiment, the invention provides compositions and methods forthe prevention of humoral rejection in a human transplant recipientwherein the transplant recipient is identified as a patient or patientpopulation at increased risk for developing a humoral rejection. Suchpatients may also be referred to as “sensitized.” The criteria for theidentification of sensitized patients is known to the skilledpractitioner. Such criteria may include, for example, patients havingdetectable levels of circulating antibodies against HLA antigens, e.g.,anti-HLA alloantibodies. Such criteria may also include patients whohave undergone previous transplantations, a pregnancy, or multiple bloodtransfusions. Patients who are at an increased risk for humoralrejection also include those having imperfect donor-recipient HLAmatching, and those transplantations which are ABO-incompatible.Sensitized individuals are candidates for pretreatment or conditioningprior to transplantation. Sensitized individuals are also candidates forpost-transplantation maintenance regimens for the prevention of humoralrejection.

In one embodiment, antibodies, compositions, and methods of theinvention comprise or are used in combination with a therapeutic regimenfor the treatment of an acute or chronic rejection. In particularembodiments, the rejection is characterized as a Stage I, a Stage II, aStage III, or a Stage IV humoral rejection.

In one embodiment, antibodies, compositions, and methods of theinvention comprise or are used in combination with a therapeutic regimenfor the treatment of an early stage humoral rejection. In particularembodiments, the early stage humoral rejection is a Stage I, II, or IIIrejection. Clinical indications of an early stage humoral rejection aredetermined according to the knowledge and skill in the art and mayinclude, for example, the development in the patient of circulatingdonor-specific anti-HLA antibodies, the presence of complement markersof antibody activity such as C4d and C3d deposits in graft biopsies, andthe presence of anti-HLA antibodies in graft biopsies. Other indicatorsof an early stage humoral rejection are known to the skilled practionerand may include, for example, the development of antiendothelialantibodies, especially antivimentin antibodies, and the development ofnonclassical MHC class I-related chain A (MICA) alloantibodies.

In one embodiment, compositions and methods of the invention comprise orare used in combination with a therapeutic regimen for the treatment ofhumoral rejection characterized in part by graft dysfunction. Inparticular embodiments, the patient or patient population in need oftreatment for humoral rejection is identified according to criteriaknown in the art for graft dysfunction. Examples of such criteria forparticular types of grafts are provided in the sections that follow. Inother embodiments, the patient or patient population in need oftreatment for humoral rejection is identified according to othercriteria that are particular to the type of tissue graft, such ashistological criteria. Examples of such criteria are also provided inthe sections that follow.

5.23.2. Bone Marrow Transplants

Compositions and methods of the invention are useful for treating orpreventing GVHD, humoral rejection, and post-transplantlymphoproliferative disorder in a bone marrow transplant recipient. Inone embodiment, compositions and methods of the invention comprise orare used in combination with a pre-transplant conditioning regimen.

In one embodiment, compositions and methods of the invention are used todeplete B cells from a bone marrow graft prior to transplantation. Thegraft may be from any suitable source, for example, cord blood stemcells, peripheral blood stem cells, or a bone marrow tap. Peripheralblood stem cells may be harvested from donor blood following a suitableconditioning regimen. Suitable regimens are known in the art and mayinclude, for example, administration of one or more of the following tothe donor prior to harvesting the donor blood: NEUPOGEN, cytokincs suchas GM-CSF, low dose chemotherapeutic regimens, and chemokine therapy.The graft may be either allogeneic or autologous to the transplantrecipient. The graft may also be a xenograft.

Compositions and methods may be useful in a number of contexts in whichthere is a hematopoietic indication for bone marrow transplantation. Inone embodiment, an autologous bone marrow graft is indicated for a Bcell leukemia or lymphoma, including, but not limited to acutelymphoblastic leukemia (“ALL”) or non-Hodgkins lymphoma, andcompositions and methods of the invention may be used for the depletionof residual malignant cells contaminating the graft. In one embodiment,an autologous bone marrow transplant is indicated for patients unable toclear a viral infection, for example a viral infection associated withEpstein Barr virus (EBV), human immunodeficiency virus (HIV), orcytomegalovirus (CMV), and compositions and methods of the invention maybe used to deplete the graft of B cells which may harbor the virus. Inanother embodiment, the graft is an allogeneic graft and compositionsand methods of the invention may be used for depleting donor B cellsfrom the graft as prophylaxis against GVHD.

In one embodiment, the indication is a B cell associated autoimmunecondition and compositions and methods of the invention may be used todeplete the deleterious B cells from the patient without the need forchemotherapy or radiation therapy conditioning regimens. In oneembodiment, compositions of the invention are administered incombination with a chemotherapy or radiation therapy regimen, whichregimen comprises a lower dose of one or more chemotherapeutic agents,or a lower dose of radiation, than the dose that is administered in theabsence of compositions of the invention. In one embodiment, the patientreceives an autologous bone marrow graft subsequent to chemotherapy orradiation therapy, wherein the graft is depleted of deleterious B cellsprior to transplantation using the compositions and methods describedherein.

A patient or patient population in need of, or likely to benefit from, abone marrow transplant is identified according to the knowledge andskill in the art. Examples of patients that may be candidates for bonemarrow transplantation include patients who have undergone chemotherapyor radiation therapy for the treatment of a cancer or an autoimmuncdisease or disorder, and patients who are unable to clear a viralinfection residing in cells of the immune system.

5.23.3. Liver Transplants

Compositions and methods of the invention are useful for treating orpreventing GVHD, humoral rejection, and post-transplantlymphoproliferative disorder in a liver transplant recipient. Inparticular embodiments, the rejection is an acute or a chronicrejection. In one embodiment, compositions and methods of the inventionare used for the prevention of GVHD, humoral rejection, andpost-transplant lymphoproliferative disorder in a liver transplantrecipient. In one embodiment, compositions and methods of the inventioncomprise or are used in combination with a pre-transplant conditioningregimen. In one embodiment, compositions of the invention areadministered to the transplant recipient. In one embodiment,compositions of the invention are contacted with the graft, ex vivo,prior to transplantation.

The liver transplant may be from any suitable source as determinedaccording to the knowledge and skill in the art. In one embodiment, theliver is an HLA-matched al logeneic graft. In another embodiment, theliver is a xenograft from a pig donor. In one embodiment, the liver isused ex vivo to filter the patient's blood, e.g., extracorporealperfusion. Extracorporeal perfusion is a form of liver dialysis in whichthe patient is surgically connected to a liver maintained outside thebody. This procedure is sometimes referred to as “bioartificial liver.”In accordance with this embodiment, compositions and methods of theinvention are used to prevent the development of antibodies againstliver antigens which may contaminate the patient's blood.

In one embodiment, compositions and methods of the invention comprise animproved therapeutic regimen for the treatment and prevention of GVHD,humoral rejection, and post-transplant lymphoproliferative disorder. Ina particular embodiment, compositions and methods of the inventioncomprise an improved therapeutic regimen, wherein the improvement liesin a decreased incidence and/or severity of complications associatedwith traditional immunosuppressive agents. In one embodiment, theincidence and/or severity of nephrotoxicity, hepatotoxicity, andhirsutism is reduced compared with traditional regimens relying oncyclosporin A or other calcinuerin inhibitors. In one embodiment, theincidence and/or severity of obesity, osteodystrophy, diabetes mellitusand susceptibility to bacterial and viral infections is reduced comparedwith traditional regimens relying on corticosteroids.

In one embodiment, compositions and methods of the invention are used incombination with lower doses of one or more traditionalimmunosuppressive agents than the doses that are used in the absence ofanti-lymphocyte antibody therapy. The lower doses may result in adecreased incidence and/or severity of one or more complicationsassociated with the one or more traditional immunosuppressive agents.

A patient or patient population in need of, or likely to benefit from, aliver transplant is identified according to the knowledge and skill inthe art. Examples of patients that may be candidates for livertransplantation include persons having one or more of the followingconditions, diseases, or disorders: acute liver failure, amyloidosis,bilirubin excretion disorders, biliary atresia, Budd-Chiari syndrome,chronic active autoimmune hepatitis, cirrhosis (either associated withviral hepatitis including hepatitis B and hepatitis C, alcoholiccirrhosis, or primary biliary cirrhosis), cholangitis, congenital factorVIII or IX disorder, copper metabolism disorders, cystic fibrosis,glycogenesis, hypercholesterolemia, lipidoses, mucopolysaccharidosis,primary sclerosing cholangitis, porphyrin metabolism disorders, purineand pyrimidine metabolism disorders, and primary benign and malignantneoplasms, especially of the liver and intrahepatic bile ducts, biliarysystem, biliary passages, or digestive system.

The clinical criteria for the identification of a patient or patientpopulation in need of, or likely to benefit from, a liver transplant canbe determined according to the knowledge and skill in the art. Suchcriteria may include, for example, one or more of the followingsymptoms: fatigue, weight loss, upper abdominal pain, purities,jaundice, liver enlargement, discolored urine, elevated alkalinephosphatase, and gamma glutamylpeptidase activity, elevated bilirubinlevels, decreased serum albumin, elevated liver-specific enzymes, lowbile production, increased blood urea nitrogen, increased creatinineand/or presence of anti-neutrophil cytoplasmic antibodies (ANCA) titers,recurrent variceal hemorrhage, intractable ascites, spontaneousbacterial peritonitis, refractory encephalopathy, severe jaundice,exacerbated synthetic dysfunction, sudden physiologic deterioration, andfulminant hepatic failure.

5.23.4. Kidney (Renal) Transplants

Compositions and methods of the invention are useful for treating orpreventing GVHD, humoral rejection, and post-transplantlymphoproliferative disorder in a renal transplant recipient. As usedherein, the term “renal transplant” encompasses the transplant of akidney and the combined transplant of a kidney and a pancreas. Inparticular embodiments, the rejection is characterized as an acuterejection or a chronic rejection.

In one embodiment, compositions and methods of the invention comprise orare used in combination with a pre-transplant conditioning regimen. Inone embodiment, a single dose of one or more of the compositions of thepresent invention is effective to reduce panel reactive antibodies anddeplete B cells in the patient or patient population. In anotherembodiment, multiple doses of one or more of the compositions of theinvention are effective to reduce panel reactive antibodies and depleteB cells in the patient or patient population. In one embodiment, asingle dose of one or more of the compositions of the present inventionis administered in combination with one or more immunosuppressive agentsand is effective to reduce panel reactive antibodies and deplete B cellsin the patient or patient population.

In certain embodiments, compositions and methods of the invention arefor treating or preventing GVHD and graft rejection in a patient havingreceived a renal transplant. In one embodiment, the patient has not yetexhibited clinical signs of rejection. In a related embodiment,compositions and methods of the invention comprise or are used incombination with a maintenance regimen for the prevention of graftrejection in the transplant recipient. In one embodiment, compositionsand methods of the invention are for the treatment of a subclinicalhumoral rejection. In a related embodiment, the patient or patientpopulation in need of treatment for a subclinical humoral rejection isindicated by the detection of Cd4 deposition in a biopsy from the graftor by the detection of circulating anti-HLA antibodies.

In one embodiment, compositions and methods of the invention comprise orare used in combination with a therapeutic regimen for the treatment ofan acute or chronic rejection episode in a transplant recipient. In oneembodiment, the patient or patient population in need of treatment foran acute or chronic rejection episode is identified by the detection ofone or more clinical indicators of rejection. In specific embodiments,the one or more clinical indicators of rejection are detected one to sixweeks post-transplantation. In one embodiment, the one or more clinicalindicators of rejection are detected 6, 12, 18, 24, 36, 48, or 60 monthspost-transplantation. In one embodiment, the acute rejection isbiopsy-confirmed acute humoral rejection.

In one embodiment, one or more of the compositions of the inventioncomprise a therapeutic regimen for the treatment of acute rejection. Ina particular embodiment, the therapeutic regimen further comprises oneor more of the following: plasmapheresis, tacrolimus/mycophenolate,intravenous immunoglobulin, immunoadsorption with protein A, andanti-CD20 antibody. In one embodiment, the patient has been on animmunosuppressive protocol prior to the development of the rejection. Ina particular embodiment, the immunosuppressive protocol includes one ormore of cyclosporine, azathioprine, and steroid therapy.

Clinical indicators of acute humoral rejection are known in the art andinclude, for example, a sudden severe deterioration of renal function,the development of oliguria, and compromised renal perfusion. Additionalindicators include, for example, inflammatory cells in peritubularcapillaries on biopsy and circulating donor-specific alloantibodies. Inone embodiment, the patient presents with one or more of the followingdiagnostic criteria for a humoral rejection of a renal allograft: (1)morphological evidence of acute tissue injury; (2) evidence of antibodyaction, such as C4d deposits or immunoglobulin and complement inarterial fibrinoid necrosis; and (3) detectable circulating antibodiesagainst donor HLA antigens or donor endothelial antigens. In oneembodiment, the patient presents with all three of the above diagnosticcriteria.

In one embodiment, the patient presents with one or more of theforegoing diagnostic criteria of acute humoral rejection andcompositions of the present invention are used in combination with oneor more of the following immunosuppressive agents to treat the acutehumoral rejection: intravenous immunoglobulin, anti-thymocyte globulins,anti-CD20 antibody, mycophenolate mofetil, or tacrolimus. In anotherembodiment, compositions of the invention are used in combination withone or more immunosuppressive agents and a procedure for the removal ofalloantibodies from the patient, such as plasmapheresis orimmunoadsorption.

In one embodiment, compositions and methods of the invention comprise orare used in combination with a therapeutic regimen for the treatment ofa chronic renal allograft rejection. In one embodiment, one or more ofthe compositions of the invention are used alone or in combination withone or more immunosuppressive agents, including for example, anti-CD154(CD40L), tacrolimus, sirolimus, and mizoribin. In one embodiment, one ormore of the anti-CD19 antibodies are used in combination with tacrolimusand mycophenolate.

Clinical indicators of chronic rejection in the kidneys are known in theart and may include, for example, arterial intimal fibrosis with intimalmononuclear cells (chronic allograft vasculopathy), duplication of theglomerular basement membranes (chronic allograft glomerulopathy),lamination of the peritubular basement membrane, C4d in peritubularcapillaries, and detectable circulating donor HLA-reactive antibodies.In one embodiment, compositions and methods of the invention comprise orare used in combination with a therapeutic regimen to treat chronicrejection before graft lesions develop.

In another embodiment, the patient or patient population in need oftreatment is identified as having one or more clinical indicators oftransplant glomerulopathy. In a related embodiment, compositions of theinvention comprise or are used in combination with a therapeutic regimencomprising one or more therapeutic agents. In certain embodiments, thetherapeutic regimen is effective to stabilize renal function and inhibitgraft rejection. In a particular embodiment, the one or more therapeuticagents include angiotensin converting enzyme (ACE) inhibitors and/orreceptor antagonists, intravenous immunoglobulin, anti-thymocyteglobulins, anti-CD20 antibody, mycophenolate mofetil, or tacrolimus.Anti-CD19 antibodies may be used in combination with mycophenolatemofetil and tacrolimus, with or without other therapeutic agents.Plasmapheresis may also be used as part of the therapeutic regimen.

A patient or patient population in need of, or likely to benefit from, arenal transplant is identified according to the knowledge and skill inthe art. Examples of patients that may be candidates for renaltransplantation include patients diagnosed with amyloidosis, diabetes(type I or type II), glomerular disease (e.g., glomerulonephritis),gout, hemolytic uremic syndrome, HIV, hereditary kidney disease (e.g.,polycystic kidney disease, congenital obstructive uropathy, cystinosis,or prune bell syndrome), other kidney disease (e.g., acquiredobstructive nephropathy, acute tubular necrosis, acute intersititialnephritis), rheumatoid arthritis, systemic lupus erythematosus, orsickle cell anemia. Other candidates for renal transplant includepatients having insulin deficiency, high blood pressure, severe injuryor burns, major surgery, heart disease or heart attack, liver disease orliver failure, vascular disease (e.g., progressive systemic sclerosis,renal artery thrombosis, scleroderma), vesicoureteral reflux, andcertain cancers (e.g., incidental carcinoma, lymphoma, multiple myeloma,renal cell carcinoma, Wilms tumor). Other candidates for renaltransplant may include, for example, heroin users, persons who haverejected a previous kidney or pancreas graft, and persons undergoing atherapeutic regimen comprising antibiotics, cyclosporin, orchemotherapy.

The clinical criteria for the identification of a patient or patientpopulation in need of, or likely to benefit from, a kidney transplantcan be determined according to the knowledge and skill in the art. Suchcriteria may include, for example, one or more of the following: urinaryproblems, bleeding, easy bruising, fatigue, confusion, nausea andvomiting, loss of appetite, pale skin (from anemia), pain in themuscles, joints, flanks, and chest, bone pain or fractures, and itching.

5.23.5. Cardiac Transplants

Compositions and methods of the invention are useful for treating orpreventing GVHD, humoral rejection, and post-transplantlymphoproliferative disorder in a cardiac transplant recipient. Inparticular embodiments, the rejection is an acute or a chronicrejection. In one embodiment, compositions and methods of the inventioncomprise or are used in combination with a pre-transplant conditioningregimen.

In certain embodiments, compositions and methods of the inventioncomprise or are used in combination with a therapeutic regimen for thetreatment of acute humoral rejection in a cardiac transplant recipient.In a particular embodiment, the therapeutic regimen further comprisesone or more of the following: plasmapheresis, intravenousimmunoglobulin, and anti-CD20 antibody therapy. The patient or patientpopulation in need of treatment for an acute humoral rejection isidentified by the detection of one or more of the clinical indicationsof acute humoral rejection. Examples of clinical indicators of acutehumoral rejection may include one or more of the following: hemodynamicdysfunction, defined by shock, hypotension, decreased cardiac output,and a rise in capillary wedge or pulmonary artery pressure. In aparticular embodiment, the acute humoral rejection is diagnosed within6, 12, 18, 24, 36, 48, or 60 months post-transplantation.

In one embodiment, compositions and methods of the invention comprise orare used in combination with a therapeutic regimen for the prevention ofrejection in a cardiac transplant recipient. In one embodiment, thetransplant recipient in need of prophylaxis against rejection isidentified as a patient or patient population having one or more of thefollowing risk factors: female sex, cytomegalovirus seropositivity,elevated response to panel reactive antibodies, positive pre- and/orpost-transplant crossmatch, and presensitization with immunosuppressiveagents.

In one embodiment, compositions and methods of the invention are for thetreatment or prevention of graft deterioration in a heart transplantrecipient. In one embodiment, the transplant recipient in need oftreatment for, or prophylaxis against, graft deterioration is identifiedas a patient or patient population having one or more of the followingclinical indications of humoral rejection: deposition of immunoglobulin,C 1 q, C3, and/or C4d in capillaries, evidence of CD68-positive cellswithin capillaries, and evidence of infiltration of the graft byinflammatory cells upon biopsy. In one embodiment, compositions of thepresent invention are used in combination with one or more of thefollowing immunosuppressive agents to treat graft deterioration in aheart transplant recipient: intravenous immunoglobulin, anti-thymocyteglobulins, anti-CD20 antibody, mycophenolate mofetil, or tacrolimus. Inanother embodiment, anti-CD19 antibody compositions may be used incombination with one or more immunosuppressive agents and a procedurefor the removal of alloantibodies from the patient, such asplasmapheresis or immunoadsorption.

In one embodiment, compositions and methods of the invention comprise orare used in combination with a therapeutic regimen for the treatment ofchronic cardiac rejection, for example chronic allograft vasculopathy,also referred to as transplant coronary artery disease. In anotherembodiment, compositions and methods of the invention comprise or areused in combination with a therapeutic regimen for the prevention oftransplant coronary artery disease in a patient or patient population atrisk. The criteria for identifying a patient or patient population atrisk of developing transplant coronary artery disease are known in theart and may include, for example, patients having poorly matchedtransplants, patients who develop circulating anti-HLA antibodies, andpatients who develop one or more clinical indications of humoralrejection early after cardiac transplant.

A patient or patient population in need of, or likely to benefit from, aheart transplant is identified according to the knowledge and skill inthe art. Examples of patients that may be candidates for hearttransplantation include those who have been diagnosed with any of thefollowing diseases and disorders: coronary artery disease,cardiomyopathy (noninflammatory disease of the heart), heart valvedisease with congestive heart failure, life-threatening abnormal heartrhythms that do not respond to other therapy, idiopathic cardiomyopathy,ischemic cardiomyopathy, dilated cardiomyopathy, ischemiccardiomyopathy, and congenital heart disease for which no conventionaltherapy exists or for which conventional therapy has failed.

The clinical criteria for the identification of a patient or patientpopulation in need of, or likely to benefit from, a heart transplant canbe determined according to the knowledge and skill in the art. Suchcriteria may include, for example, one or more of the following:ejection fraction less than 25%, intractable angina or malignant cardiacarrhythmias unresponsive to conventional therapy, and pulmonary vascularresistance of less than 2 Wood units. In addition, the patient orpatient population in need of a heart transplant may be identified byperforming a series of tests according to the knowledge and skill in theart. Such tests include, for example, resting and stressechocardiograms, EKG, assay of blood creatinine levels, coronaryarteriography, and cardiopulmonary evaluation including right- andleft-heart catheterization.

5.23.6. Lung Transplant

Compositions and methods of the invention are useful for treating orpreventing GVHD, humoral rejection, and post-transplantlymphoproliferative disorder in a lung transplant recipient. Inparticular embodiments, the rejection is characterized as an acute or achronic rejection. In one embodiment, compositions and methods of theinvention comprise or are used in combination with a pre-transplantconditioning regimen.

A patient or patient population in need of, or likely to benefit from, alung transplant is identified according to the knowledge and skill inthe art. Examples of patients that may be candidates for lungtransplantation include patients having one of the following diseases orconditions: bronchiectasis, chronic obstructive pulmonary disease,cystic fibrosis, Eisenmenger syndrome or congenital heart disease withEisenmenger syndrome. emphysema, eosinophilic granuloma of the lung, orhistiocytosis X, inhalation/burn trauma, lymphangioleiomyomatosis (LAM),primary pulmonary hypertension, pulmonary fibrosis (scarring of thelung), or sarcoidosis.

The clinical criteria for the identification of a patient or patientpopulation in need of, or likely to benefit from, a lung transplant canbe determined according to the knowledge and skill in the art. Suchcriteria may include, for example, one or more of the following: Chronicobstructive pulmonary disease (COPD) and alpha1-antitrypsin deficiencyemphysema characterized by one or more of the following indicators:postbronchodilator FEV1 of less than 25% predicted, resting hypoxemia,i.e., PaO₂ of less than 55-60 mm Hg, hypercapnia. secondary pulmonaryhypertension, a rapid rate of decline in FEV1, or life-threateningexacerbations; cystic fibrosis characterized by one or more of thefollowing indicators: postbronchodilator FEV1 of less than 30%predicted, resting hypoxemia, hypercapnia, or increasing frequency andseverity of exacerbations; idiopathic pulmonary fibrosis characterizedby one or more of the following indicators: vital capacity (VC) and TLCof less than 60-65% predicted, and resting hypoxemia; secondarypulmonary hypertension characterized by clinical, radiographic, orphysiologic progression while on medical therapy; primary pulmonaryhypertension characterized by one or more of the following indicators:NYHA functional class III or IV, mean right atrial pressure of greaterthan 10 mm Hg, mean pulmonary arterial pressure of greater than 50 mmHg, cardiac index of less than 2.5 L/min/m², and failure of therapy withlong-term prostacyclin infusion.

5.23.7. Post-Transplant Lymphoproliferative Disorder

The immunosuppression necessary for successful transplantation can giverise to a post-transplant lymphoproliferative disorder of B cell origin.Generally, a post-transplant lymphoproliferative disorder is associatedwith Epstein-Barr virus infected cells. Post-transplantlymphoproliferative disorder (PTLD) can range in severity from a benignself-limiting mononucleosis-like syndrome to an aggressive non-Hodgkinslymphoma. Compositions and methods of the present invention may be usedto treat PTLD arising from any transplant. The transplant may be a solidorgan transplant, for example a heart transplant, a liver transplant, akidney transplant, or a combined kidney-pancreas transplant. In oneembodiment, compositions and methods of the invention are used to treatPTLD as part of a therapeutic regimen that includes a temporarycessation or reduction of other immunosuppressive therapy.

In one embodiment, anti-CD19 antibody compositions are administered aspart of a therapeutic regimen including one or more of the following:high dose intravenous gamma globulin, a cytokine, an anti-viral agent,and an anti-CD20 monoclonal antibody. The therapeutic regimen mayinclude a temporary cessation or reduction of immunosuppression therapy.In one embodiment, intravenous gamma globulin is administered at a dailydose of 0.4 g/kg for 1 to 5 days, preferably for 3 days, and thecytokine is interferon alpha administered for at least 7 days. In oneembodiment, one or more cytokines is used in the regimen. In oneembodiment, one or more anti-viral agents is used in the regimen. Theanti-viral agent may be selected from any suitable anti-viral agentknown to those of skill in the art. In one embodiment, the anti-viralagent is aciclovir or ganciclovir. The anti-viral agent may beadministered for at least one or two weeks. The anti-viral agent mayalso be administered for longer periods, for example, 1 month, 2 months,3 months, 4 months, or 5 months.

5.24. Patient Diagnosis and Therapeutic Regimens: Autoimmune Disease

According to certain aspects of the invention, the treatment regimen anddose used with compositions and methods of the invention is chosen basedon a number of factors including, but not limited to, the stage of theautoimmune disease or disorder being treated. Appropriate treatmentregimens can be determined by one of skill in the art for particularstages of an autoimmune disease or disorder in a patient or patientpopulation. Dose response curves can be generated using standardprotocols in the art in order to determine the effective amount ofcompositions of the invention for treating patients having differentstages of a autoimmune disease or disorder. In general, patients havingmore activity of a autoimmune disease or disorder will require higherdoses and/or more frequent doses which may be administered over longerperiods of time in comparison to patients having less activity of anautoimmune disease or disorder.

Anti-CD19 antibodies, compositions and methods described herein may bepracticed to treat an autoimmune disease or disorder. The term“autoimmune disease or disorder” refers to a condition in a subjectcharacterized by cellular, tissue and/or organ injury caused by animmunologic reaction of the subject to its own cells, tissues and/ororgans. The term “inflammatory disease” is used interchangeably with theterm “inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, including, but not limited to chronicinflammation. Autoimmune disorders may or may not be associated withinflammation. Moreover, inflammation may or may not be caused by anautoimmune disorder. Thus, certain disorders may be characterized asboth autoimmune and inflammatory disorders. Exemplary autoimmunediseases or disorders include, but are not limited to: alopecia areata,ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sdisease, autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune oophoritis and orchitis,autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid,cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immunedysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, diabetes, eosinophilic fascites,fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,Guillain-Barre, Hashimoto's thyroiditis, Henoch-Schonlein purpura,idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Meniere's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus-related disorders (e.g., pemphigusvulgaris), pernicious anemia, polyarteritis nodosa, polychrondritis,polyglandular syndromes, polymyalgia rheumatica, polymyositis anddermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis,psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter'ssyndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sji5gren'ssyndrome, stiff-man syndrome, systemic lupus erythematosis (SLE),Sweet's syndrome, Still's disease, lupus erythematosus, takayasuarteritis, temporal arteristis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, graft versus host disease,urticaria, Vogt-Koyanagi-Hareda syndrome and chronic inflammationresulting from chronic viral or bacteria infections.

Anti-CD19 immunotherapy encompasses the administration of an anti-CD19antibody as a single agent therapeutic for the treatment of anautoimmune disease or disorder. In one embodiment, an anti-CD19immunotherapy of the invention encompasses the administration of ananti-CD19 antibody capable of inhibiting in vitro stimulated B cellproliferation. In another embodiment, an anti-CD19 immunotherapy of theinvention encompasses the administration of an Fc variant anti-CD19antibody wherein said Fc variant has altered binding affinity to one ormore Fc ligand relative to a comparable non-variant molecule. In aspecific embodiment, an anti-CD19 immunotherapy of the inventionencompasses the administration of an Fc variant anti-CD19 antibodywherein said Fc variant has enhanced binding to Fc gamma receptor IIBrelative to a comparable non-variant Fc domain.

Anti-CD19 immunotherapy further encompasses the administration of ananti-CD19 bispecific antibody as a single agent therapeutic for thetreatment of an autoimmune disease or disorder. In one embodiment, ananti-CD19 immunotherapy of the invention encompasses the administrationof an anti-CD19 bispecific antibody capable to specifically bind to afirst and second antigen, wherein said first antigen is human CD19 andsaid second antigen is an Fc gamma receptor selected from the groupconsisting of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and/or FcγRIV. In afurther embodiment, an anti-CD19 immunotherapy of the inventionencompasses the administration of an anti-CD19 bispecific antibodycapable of specifically binding to human CD19 and FcγRIIB

CD19 is expressed on immature B cells, therefore an anti-CD19 mAb may beparticularly suited for depleting pre-B cells and immature B cells, e.g,in the bone marrow.

5.24.1. Diagnosis of Autoimmune Diseases or Disorders

The diagnosis of an autoimmunc disease or disorder is complicated inthat each type of autoimmune disease or disorder manifests differentlyamong patients. This heterogeneity of symptoms means that multiplefactors are typically used to arrive at a clinical diagnosis. Generally,clinicians use factors, such as, but not limited to, the presence ofautoantibodies, elevated cytokine levels, specific organ dysfunction,skin rashes, joint swelling, pain, bone remodeling, and/or loss ofmovement as primarily indicators of an autoimmune disease or disorder.For certain autoimmune diseases or disorders, such as RA and SLE,standards for diagnosis are known in the art. For certain autoimmunediseases or disorders, stages of disease have been characterized and arewell known in the art. These art recognized methods for diagnosingautoimmune diseases and disorders as well as stages of disease andscales of activity and/or severity of disease that are well known in theart can be used to identify patients and patient populations in need oftreatment for an autoimmune disease or disorder using compositions andmethods of the invention.

5.24.2. Clinical Criteria for Diagnosing Autoimmune Diseases orDisorders

Diagnostic criteria for different autoimmune diseases or disorders areknown in the art. Historically, diagnosis is typically based on acombination of physical symptoms. More recently, molecular techniquessuch as gene-expression profiling have been applied to develop moleculardefinitions of autoimmunc diseases or disorders. Exemplary methods forclinical diagnosis of particular autoimmune diseases or disorders areprovided below. Other suitable methods will be apparent to those skilledin the art.

In certain embodiments, patients with low levels of autoimmune diseaseactivity or patients with an early stage of an autoimmune disease (fordiseases where stages are recognized) can be identified for treatmentusing anti-CD19 antibody compositions and methods. The early diagnosisof autoimmune disease is difficult due to the general symptoms andoverlap of symptoms among diseases. In such embodiments, a patienttreated at an early stage or with low levels of an autoimmune diseaseactivity has symptoms comprising at least one symptom of an autoimmunedisease or disorder. In related embodiments, a patient treated at anearly stage or with low levels of an autoimmune disease has symptomscomprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15symptoms of an autoimmune disease or disorder. The symptoms may be ofany autoimmune diseases and disorders or a combination thereof. Examplesof autoimmune disease and disorder symptoms are described below.

5.24.3. Rheumatoid Arthritis

Rheumatoid arthritis is a chronic disease, mainly characterized byinflammation of the lining, or synovium, of the joints. It can lead tolong-term joint damage, resulting in chronic pain, loss of function anddisability. Identifying patients or patient populations in need oftreatment for rheumatoid arthritis is a process. There is no definitivetest that provides a positive or negative diagnosis of rheumatoidarthritis. Clinicians rely on a number of tools including, medicalhistories, physical exams, lab tests, and X-rays.

Physical symptoms vary widely among patients and commonly include, butare not limited to, joint swelling, joint tenderness, loss of motion injoints, joint malalignment, bone remodeling, fatigue, stiffness(particularly in the morning and when sitting for long periods of time),weakness, flu-like symptoms (including a low-grade fever), painassociated with prolonged sitting, the occurrence of flares of diseaseactivity followed by remission or disease inactivity, rheumatoid nodulesor lumps of tissue under the skin (typically found on the elbows, theycan indicate more severe disease activity), muscle pain, loss ofappetite, depression, weight loss, anemia, cold and/or sweaty hands andfeet, and involvement of the glands around the eyes and mouth, causingdecreased production of tears and saliva (Sjogren's syndrome). ForSjogren's specifically, the following references may be used, Fox et al.Arthritis Rheum. (1986) 29:577-586, and Vitali et al. Ann. Rheum. Dis.(2002). 61:554-558.

Apart form physical symptoms, clinicians commonly use tests, such as,but not limited to, complete blood count, erythrocyte sedimentation rate(ESR or sed rate), C-reactive protein, rheumatoid factor, anti-DNAantibodies, antinuclear antibodies (ANA), anti-cardiolipin antibodies,imaging studies, radiographs (X-rays), magnetic resonance imaging (MRI)of joints or organs, joint ultrasound, bone scans, and bone densitometry(DEXA). These tests are examples of tests that can be used inconjunction with compositions and methods of the invention to check forabnormalities that might exist (i.e., identify patients or patientpopulations in need of treatment) or to monitor side effects of drugsand check progress.

Early symptoms of rheumatoid arthritis commonly are found in the smallerjoints of the fingers, hands and wrists. Joint involvement is usuallysymmetrical, meaning that if a joint hurts on the left hand, the samejoint will hurt on the right hand. In general, more joint erosionindicates more severe disease activity.

Symptoms of more advanced disease activity include damage to cartilage,tendons, ligaments and bone, which causes deformity and instability inthe joints. The damage can lead to limited range of motion, resulting indaily tasks (grasping a fork, combing hair, buttoning a shirt) becomingmore difficult. Skin ulcers, greater susceptibility to infection, and ageneral decline in health are also indicators of more advanced diseaseactivity.

Progression of rheumatoid arthritis is commonly divided into threestages. The first stage is the swelling of the synovial lining, causingpain, warmth, stiffness, redness and swelling around the joint. Secondis the rapid division and growth of cells, or pannus, which causes thesynovium to thicken. In the third stage, the inflamed cells releaseenzymes that may digest bone and cartilage, often causing the involvedjoint to lose its shape and alignment, more pain, and loss of movement.

Molecular techniques can also be used to to identify patients or patientpopulations in need of treatment. For example, rheumatoid arthritis hasbeen shown to be associated with allelic polymorphisms of the humanleukocyte antigen (HLA)-DR4 and HLA-DRB1 genes (Ollier and Winchester,1999, Genes and Genetics of Autoimmunity. Basel, Switzerland; Stastny,1978, N. Engl J Med 298:869-871; and Gregersen et al., 1987, ArthritisRheum 30:1205-1213). Rheumatoid arthritis patients frequently expresstwo disease-associated HLA-DRB1*04 alleles (Weyand et al., 1992 AnnIntern Med 117:801-806). Patients can be tested for allelicpolymorphisms using methods standard in the art. MHC genes are not theonly germline-encoded genes influencing susceptibility to RA that can beused to diagnose or identify patients or patient populations in need oftreatment. Female sex clearly increases the risk, and female patientsdevelop a different phenotype of the disease than do male patients. Anymolecular indicators of rheumatoid arthritis can be used to identifypatients or patient populations in need of treatment with an anti-CD19antibody composition or method.

Methods for determining activity of rheumatoid arthritis in a patient inrelation to a scale of activity are well known in the art and can beused in connection with pharmaceutical compositions and methods of theinvention. For example, the American College of Rheumatologists Score(ACR score) can be used to determine the activity of rheumatoidarthritis of a patient or a patient population. According to thismethod, patients are given a score that correlates to improvement. Forexample, patients with a 20% improvement in factors defined by the ACRwould be given an ACR20 score.

Initially, a patient exhibiting the symptoms of rheumatoid arthritis maybe treated with an analgesic. In other embodiments, a patient diagnosedwith or exhibiting the symptoms of rheumatoid arthritis is initiallytreated with nonsteroidal anti-inflammatory (NSAID) compounds. As thedisease progresses and/or the symptoms increase in severity, rheumatoidarthritis may be treated by the administration of steroids such as butnot limited to dexamethasone and prednisone. In more severe cases, achemotherapeutic agent, such as but not limited to methotrexate orcytoxin may be administered to relieve the symptoms of rheumatoidarthritis.

In certain instances, rheumatoid arthritis may be treated byadministration of gold, while in other instances a biologic, such as anantibody or a receptor (or receptor analog) may be administered.Examples of such therapeutic antibodies are RITUXAN™ (rituximab) andREMICADE® (infliximab). An illustrative example of a soluble receptorthat can be administered to treat rheumatoid arthritis is ENBREL®(etanercept).

In extremely severe cases of rheumatoid arthritis, surgery may beindicated. Surgical appoaches may include, but not be limited to:synovectomy to reduce the amount of inflammatory tissue by removing thediseased synovium or lining of the joint; arthroscopic surgery to taketissue samples, remove loose cartilage, repair tears, smooth a roughsurface or remove diseased synovial tissue; osteotomy, meaning “to cutbone,” this procedure is used to increase stability by redistributingthe weight on the joint; joint replacement surgery or arthroplasty forthe surgical reconstruction or replacement of a joint; or arthrodesis orfusion to fuse two bones together.

In certain embodiments of the methods of invention, a patient can betreated with an anti-CD19 antibody prior, concurrent, or subsequent toany of the therapies disclosed above. Moreover, anti-CD19 antibodies ofthe present invention may be administered in combination with any of theanalgesic, NSAID, steroid, or chemotherapeutic agents noted above, aswell as in combination with a biologic administered for the treatment ofrheumatoid arthritis.

5.24.4. Systemic Lupus Erythematosis (SLE)

Systemic lupus erythematosis (SLE) is a chronic (long-lasting) rheumaticdisease which affects joints, muscles and other parts of the body.Patients or patient populations in need of treatment for SLE can beidentified by examining physical symptoms and/or laboraotry testresults. Physical symptoms vary widely among patients. For example, inSLE, typically 4 of the following 11 symptoms exist before a patient isdiagnosed with SLE: 1) malar rash: rash over the cheeks; 2) discoidrash: red raised patches; 3) photosensitivity: reaction to sunlight,resulting in the development of or increase in skin rash; 4) oralulcers: ulcers in the nose or mouth, usually painless; 5) arthritis:nonerosive arthritis involving two or more peripheral joints (arthritisin which the bones around the joints do not become destroyed); 6)serositis pleuritis or pericarditis: (inflammation of the lining of thelung or heart); 7) renal disorder: excessive protein in the urine(greater than 0.5 gm/day or 3+ on test sticks) and/or cellular casts(abnormal elements the urine, derived from red and/or white cells and/orkidney tubule cells); 8) neurologic disorder: seizures (convulsions)and/or psychosis in the absence of drugs or metabolic disturbances whichare known to cause such effects; 9) hematologic disorder: hemolyticanemia or leukopenia (white blood count below 4,000 cells per cubicmillimeter) or lymphopenia (less than 1,500 lymphocytes per cubicmillimeter) or thrombocytopenia (less than 100,000 platelets per cubicmillimeter) (The leukopenia and lymphopenia must be detected on two ormore occasions. The thrombocytopenia must be detected in the absence ofdrugs known to induce it); 10) antinuclear antibody: positive test forantinuclear antibodies (ana) in the absence of drugs known to induce it;and/or 11) immunologic disorder: positive anti-double stranded anti-DNAtest, positive anti-sm test, positive antiphospholipid antibody such asanticardiolipin, or false positive syphilis test (vdrl).

Other physical symptoms that may be indicative of SLE include, but arenot limited to, anemia, fatigue, fever, skin rash, muscle aches, nausea,vomiting and diarrhea, swollen glands, lack of appetite, sensitivity tocold (Raynaud's phenomenon), and weight loss.

Laboratory tests can also be used to to identify patients or patientpopulations in need of treatment. For example, a blood test can be usedto detect a autoantibodies found in the blood of almost all people withSLE. Such tests may include but are not limited to tests for antinuclearantibodies (ANA) in the absence of drugs known to induce it (Rahman, A.and Hiepe, F. Lupus. (2002). 11(12):770-773), anti-double strandedanti-DNA (Keren, D. F. Clin. Lab. Med. (2002) 22(2):447-474.), anti-Sm,antiphospholipid antibody such as anticardiolipin (Gezer, S. Dis. Mon.2003. 49(12):696-741), or false positive syphilis tests (VDRL).

Other tests may include a complement test (C3, C4, CH50, CH100) can beused to measure the amount of complement proteins circulating in theblood (Manzi et al. Lupus 2004. 13(5):298-303), a sedimentation rate(ESR) or C-reactive protein (CRP) may be used to measure inflammationlevels, a urine analysis can be used to detect kidney problems, chestX-rays may be taken to detect lung damage, and an EKG can be used todetect heart problems.

Chronic SLE is associated with accumulating collateral damage toinvolved organ, particularly the kidney. Accordingly, early therapeuticintervention is desirable, i.e. prior to, for example, kidney failure.Available treatments for SLE are similar to those available forrheumatoid arthritis. These include initial treatments, either with ananalgesic or a nonsteroidal anti-inflammatory (NSAID) compound. As thedisease progresses and/or the symptoms increase in severity, SLE may betreated by the administration of steroids such as but not limited todexamethasone and prednisone.

In more severe cases, a chemotherapeutic agent, such as but not limitedto methotrexate or cytoxin may be administered to relieve the symptomsof SLE. However, this approach is not preferred where the patient is afemale of child-bearing age. In such instances, those therapeuticapproaches that do not interfere with the reproductive capacity of thepatient are preferred.

In certain instances, SLE may be treated by administration of abiologic, such as an antibody or a receptor (or receptor analog).Examples of such therapeutic antibodies are RITUXAN™ (rituximab) andREMICADE® (infliximab). An illustrative example of a soluble receptorfor an inflammatory cytokine that can be administered to treat SLE isENBREL® (etanercept).

In certain embodiments of the methods of invention, a patient can betreated with an anti-CD19 antibody prior, concurrent, or subsequent toany of the therapies disclosed above that are used for the treatment ofSLE. Moreover, anti-CD19 antibodies of the present invention may beadministered in combination with any of the analgesic, NSAID, steroid,or chemotherapeutic agents noted above, as well as in combination with abiologic administered for the treatment of SLE.

5.24.5. Idiopathic/Autoimmune Thrombocytopenta Purpura (ITP)

Idiopathic/autoimmune thrombocytopenia purpura (ITP) is a disorder ofthe blood characterized by immunoglobulin G (IgG) autoantibodies thatinteract with platelet cells and result in the destruction of thoseplatelet cells. Typically, the antibodies are specific to plateletmembrane glycoproteins. The disorder may be acute (temporary, lastingless than 2 months) or chronic (persisting for longer than 6 months).Patients or patient populations in need of treatment for ITP can beidentified by examining a patient's medical history, physical symptoms,and/or laboratory test results. (Provan, D., and Newland, A., Br. J.Haematol. (2002) 118(4):933-944; George, J. N. Curr. Hematol. (2003)2(5):381-387; Karptkin, S. Autoimmunity. (2004) 37(4):363-368; Cines, D.B., and Blanchette, V. S., N. Engl. J. Med. (2002) 346(13)995-1008).

Physical symptoms include purplish-looking areas of the skin and mucousmembranes (such as the lining of the mouth) where bleeding has occurredas a result of a decrease in the number of platelet cells. The mainsymptom is bleeding, which can include bruising (“ccchymosis”) and tinyred dots on the skin or mucous membranes (“pctcchiac”). In someinstances bleeding from the nose, gums, digestive or urinary tracts mayalso occur. Rarely, bleeding within the brain occurs. Common signs,symptoms, and precipitating factors also include, but are not limitedto, abrupt onset (childhood ITP), gradual onset (adult ITP), nonpalpablepetechiae, purpura, menorrhagia, epistaxis, gingival bleeding,hemorrhagic bullae on mucous membranes, signs of GI bleeding,menometrorrhagia, evidence of intracranial hemorrhage, nonpalpablespleen, retinal hemorrhages, recent live virus immunization (childhoodITP), recent viral illness (childhood ITP), spontaneous bleeding whenplatelet count is less than 20,000/mm³, and bruising tendency.

Laboratory test that can be used to diagnose ITP include, but are notlimited to, a complete blood count test, or a bone marrow examination toverify that there are adequate platelet-forming cells (megakaryocyte) inthe marrow and to rule out other diseases such as metastatic cancer andleukemia. Isolated thrombocytopenia is the key finding regardinglaboratory evaluation. Giant platelets on peripheral smear areindicative of congenital thrombocytopenia. A CT scan of the head may bewarranted if concern exists regarding intracranial hemorrhage.

The current treatments for ITP include, platelet transfusions andsplenectomy. Other treatments include, the administration ofglucocorticoids, administration of immunosuppressive agents,administration of agents that enhance platelet production, such asIL-11, and agents that activate megakaryocytes to produce platelets,such as thrombopoietin (TPO).

In more severe cases, a chemotherapeutic agent, such as but not limitedto vincristine and vinblastine may be administered to relieve thesymptoms of ITP. However, this approach is not preferred where thepatient is a female of child-bearing age. In such instances, thosetherapeutic approaches that do not interfere with the reproductivecapacity of the patient are preferred.

In certain instances, ITP may be treated by administration of abiologic, such as an antibody or a receptor (or receptor analog).Examples of such therapeutic antibodies are anti-CD20 antibodies, suchas, Rituximab.

In certain embodiments of the methods of invention, a patient can betreated with an anti-CD19 antibody prior, concurrent, or subsequent toany of the therapies disclosed above that are used for the treatment ofITP. Moreover, anti-CD19 antibodies of the present invention may beadministered in combination with any of the agents noted above, as wellas in combination with a biologic administered for the treatment of ITP.

5.24.6. Pemphigus and Pemphigoid-Related Disorders

Both pemphigus- and pemphigoid-related disorders are a heterogenousgroup of autoimmune diseases characterized by a blistering condition ofthe skin and/or mucosal surfaces. In both diseases, the blistering iscaused by autoimmune antibodies that recognize various proteinsexpressed on the surface of epithelial cells in the dermis and/orepidermis.

In patients with pemphigus-related disease, the blistering occurs withinthe epidermis and is due to the binding of autoantibodies specific fordesmoglein 1 (Dsgl) and/or desmoglein 3 (Dsg3). The classic subtypes ofpemphigus can be distinguished according to anti-desmoglein antibodyspecificities. Patients with pemphigus foliaceus (PF) produce anti-Dsglantibodies only. Patients with pemphigus vulgaris (PV) andparaneoplastic pemphigus (PNP) produce anti-Dsg3 antibodies if theirlesions are restricted to mucosal tissues. In contrast, PV and PNPpatients with lesions of the skin and mucosa produce both anti-Dsg 1 and-Dsg3 autoantibodies. (Nagasaka, T., et al. J. Clin. Invest. 2004.114:1484-1492; Seishema, M., et al. Arch Dermatol. 2004.140(12):1500-1503; Amagai, M., J. Dermatol. Sci. 1999. 20(2):92-102)

In patients with pemphigoid-related disease including but not limitedto, bulous phemphigoid, urticarial bulous pemphigoid, cicatricialpemphigoid, epidermolysis bullosa acquisita, and Linear IgA bullousdermatosis, the blistering occurs at the interface of the dermis withthe epidermis. The most common form of pemphigoid disease is bulouspemphigoid (BP) which is characterized by the presence of autoantibodiesthat bind the bullous pemphigoid antigen 180 (BP180), bullous pemphigoidantigen 230 (BP230), laminin 5, and/or beta 4 integrin. (Fontao, L., etal. Mol. Biol. Cell. 2003) 14(5):1978-1992; Challacombe, S. J., et alActa Odontol. Scand. (2001). 59(4):226-234.)

Patients or patient populations in need of treatment for pemphigus- orpemphigoid-related disorders can be identified by examining a patient'smedical history, physical symptoms, and/or laboraotry test results(reviewed in: Mutasim, D. F. Drugs Aging. (2003).20(9):663-681; Yeh, S.W. et al. Dermatol. Ther. (2003). 16(3):214-223; Rosenkrantz, W. S. Vet.Dermatol. 15(2):90-98.).

Typically, diagnosis of these pemphigus- or pemphigoid-related disordersis made by skin biopsy. The biopsy skin sample is examinedmicroscopically to determine the anatomical site of the blister (e.g.epidermis or between dermis and epidermis). These findings arecorrelated with direct or indirect immunohistochemical analyses todetect the presence of autoantibodies at the site of the lesion. Serumsamples from patients may also be examined for the presence ofcirculating autoantibodies using an ELISA-based test for specificproteins. Several ELISA-based assays have been described for detectionof desmoglein antibodies in human samples (Hashimoto, T. Arch. Dermatol.Res. (2003) 295 Supp1.1:S2-11). The presence of these desmogleinautoantibodies in biopsy samples is diagnistic of pemphigus.

Clinically, pemphigus vulgaris can be diagnosed by the presence ofblisters in the mouth. Inflammation or erosions may also be present inthe lining of the eye and eyelids, and the membranes of the nose orgenital tract. Half of the patients also develop blisters or erosions ofthe skin, often in the groin, underarm, face, scalp and chest areas.Pemphigus foliaccus is a superficial, relatively mild form of pemphigus.It usually manifests on the face and scalp, but also involves the backand chest. Lesions do not occur in the mouth. The blisters are moreconfined to the outermost surface and often itch. Paraneoplasticpemphigus is very rare and generally occurs in people who have cancer.The lesions are painful and affect the mouth, lips and esophagus(swallowing tube) as well as the skin. Due to involvement of theairways, signs of respiratory disease may occur and can belife-threatening.

The current treatments for pemphigus or pemphigoid-related diseaseincludes the topical administration of creams and ointments to alleviatethe discomfort associated with the skin condition, the administration ofanti-inflammatory agents or the administration of immunosuppressiveagents.

In certain embodiments of the methods of invention, a patient can betreated with an anti-CD19 antibody prior, concurrent, or subsequent toany of the therapies disclosed above that are used for the treatment ofpemphigoid or pemphigoid related disease. Moreover, anti-CD19 antibodiesof the present invention may be administered in combination with any ofthe agents noted above.

5.24.7. Autoimmune Diabetes

According to certain aspects of the invention, a patient in need oftreatment for autoimmunc diabetes, also known as type 1A diabetes, canbe treated with anti-CD19 antibody compositions and methods. Type IAdiabetes is an autoimmune disease caused by the synergistic effects ofgenetic, environmental, and immunologic factors that ultimately destroythe pancreatic beta cells. The consequences of pancreatic beta celldestruction are a decrease in beta cell mass, a reduced insulinproduction/secretion and a gradual rise in blood glucose levels.

Patients or patient populations in need of treatment for type 1Adiabetes can be identified by examining a patient's medical history,physical symptoms, and/or laboratory test results. Symptoms often comeon suddenly and include, but are not limited to, low or non-existentblood insulin levels, increased thirst, increased urination, constanthunger, weight loss, blurred vision, and/or fatigue. Overt diabetes doesnot usually become evident until a majority of beta cells are destroyed(>80%). Typically, diabetes is clinically diagnosed if a patient has arandom (without regard to time since last meal) blood glucoseconcentration >11.1 mmol/L (200 mg/dL) and/or a fasting (no caloricintake for at least 8 hours) plasma glucose >7.0 mmol/L (126 mg/dl)and/or a two-hour plasma glucose ≥11.1 mmol/L (200 mg/dL). Ideally,these tests should be repeated on different days with comparable resultsbefore diagnosis is confirmed. (Harrison's Principles of InternalMedicine, 16^(th) ed./editors, Dennis L. Kasper, et al. The McGraw-HillCompanies, Inc. 2005 New York, N.Y.).

Although the precise etiology of type 1A diabetes is unknown, thereexists clear genetic linkage to specific HLA serotypes. In particular,autoimmune diabetes is associated with HLA DR3 and DR4 serotypes. Thepresence of both DR3 and DR4 confers the highest known genetic risk.Susceptibility to autoimmune diabetes is also linked to HLA class II(HLA-DQB1*0302. In contrast, HLA haplotypes with DRB1-1501 andDQA1-0102-DQB1-0602 are associated with protection from type 1A diabetes(Redondo, M. J., et al. J. Clin. Endocrinol. Metabolism (2000)10:3793-3797.)

The destruction of the insulin producing beta islet cells can beaccompanied by islet cell autoantiboides, activated lymphocyticinfiltrates in the pancreas and draining lymph nodes, T lymphocytesresponsive to islet cell proteins, and release of inflammatory cytokineswithin the islets (Harrison's Principles of Internal Medicine, 16^(th)ed./editors, Dennis L. Kasper, et al. The McGraw-Hill Companies, Inc.2005 New York, N.Y.).

Autoantibodies associated with type 1A diabetes include but are notlimited to antibodies that bind insulin, glutamic acid decarboxylase(GAD), ICA-512/IA-2, phogrin, islet ganglioside and carboxypeptidase H(Gianani, R. and Eisenbarth, G. S. Immunol. Rev. (2005) 204:232-249;Kelemen, K. et al, J. Immunol. (2004) 172(6):3955-3962); Falorni, A. andBorozzetti, A. Best Pract. Res. Clin. Endocrinol. Metab. 2005.19(1):119-133.)

The current treatments for autoimmune diabetes include theadministration of vitamin D, corticosteroids, agents which control bloodpressure and agents that control glycemia (blood sugar levels).

In certain embodiments of the methods of invention, a patient can betreated with an anti-CD19 antibody prior, concurrent, or subsequent toany of the therapies disclosed above that are used for the treatment ofautoimmune diabetes. Moreover, anti-CD19 antibodies of the presentinvention may be administered in combination with any of the agentsnoted above.

5.24.8. Systemic Sclerosis (Scleroderma) and Related Disorders

Systemic sclerosis also known as Scleroderma encompasses a heterogeneousgroup of diseases including but not limited to, Limited cutaneousdisease, Diffuse cutaneous disease, Sine scleroderma, Undifferentiatedconnective tissue disease, Overlap syndromes, Localized scleroderma,Morphea, Linear scleroderma, En coup de saber, Scleredema adultorum ofBuschke, Scleromyxedema, Chronic graft-vs.-host disease, Eosinophilicfasciitis, Digital sclerosis in diabetes, and Primary anylooidosisandanyloidosis associated with multiple myeloma. (Reviewed in: Harrison'sPrinciples of Internal Medicine, 16^(th) ed./editors, Dennis L. Kasper,et al. The McGraw-Hill Companies, Inc. 2005 New York, N.Y.).

Clinical features associated with scleroderma can include Raynaud'sphenomenon, skin thickening, subcutaneious calcinosis, telangiectasia,arthralgias/arthritis, myopathy, esophageal dysmotility. pulmonaryfibrosis, isolated pulmonary arterial hypertension, congestive heartfailure and renal crisis. The extent to which an patient displays one ormore of these disease manifestations can influence the diagnosis andpotential treatment plan.

Autoantibodies include: Anti-topioisomerase 1, anticentromere, anti-RNApolymerase I, II, and/or III, anti-Th RNP, anti-U, RNP(anti-fibrillarin), anti-PM/Sci, anti-nuclear antibodies (ANA).

Identification of patients and patient populations in need of treatmentof scleroderma can be based on clinical history and physical findings.Patients or patient populations in need of treatment for scleroderma canbe identified by examining a patient's medical history, physicalsymptoms, and/or laboraotry test results. Diagnosis may be delayed inpatients without significant skin thickening. Laboratory, X-ray,pulmonary function tests, and skin or renal (kidney) biopsies can beused to determine the extent and severity of internal organ involvement.

In the early months or years of disease onset, scleroderma may resemblemany other connective tissue diseases, such as, but not limited to,Systemic Lupus Erythcmatosus, Polymyositis, and Rheumatoid Arthritis.

The most classic symptom of systemic sclerosis (scleroderma) issclerodactyly. Initial symptoms include swollen hands, which sometimesprogress to this tapering and claw-like deformity. Not everyone withscleroderma develops this degree of skin hardening. Other symptoms caninclude morphea, linear sclerodactyly (hardened fingers), Raynaud'ssyndrome, calcinosis, and telangiectasia.

Blood tests such as antinuclear antibody (ANA) tests can be used in thediagnosis of both localized and systemic scleroderma. For example,anti-centromere antibodies (ACA) and anti-Sc1-70 antibodies areindicative of patients in need of treatment for systemic sclerosis (Hoet al., 2003, Arthritis Res Ther. 5:80-93); anti-topo II alpha antibodyare indicative of patients in need of treatment for local scleroderma;and anti-topo I alpha antibody are indicative of patients in need oftreatment for systemic scleroderma. Several types of scleroderma andmethods for diagnosing these types are recognized and well known in theart, including, but not limited to, juvenile scleroderma (Foeldvari,Curr Opin Rheumatol 14:699-703 (2002); Cefle et al., Int J Clin Pract.58:635-638 (2004)); localized scleroderma; Nodular Scleroderma (Cannick,J Rheumatol. 30:2500-2502 (2003)); and Systemic scleroderma, including,but not limited to, Calcinosis, Raynaud's, Esophagus, Sclerodactyly, andTelangiectasia (CREST), limited systemic scleroderma, and diffusesystemic scleroderma. Systemic scleroderma is also known as systemicsclerosis (SSc). It may also be referred to as Progressive SystemicSclerosis (PSSc), or Familial Progressive Systemic Sclerosis (FPSSc)(Nadashkevich et al., Med Sci Monit. 10:CR615-621 (2004); Frances etal., Rev Prat. 52:1884-90 (2002)). Systemic sclerosis is a multisystemdisorder characterized by the presence of connective tissue sclerosis,vascular abnormalities concerning small-sized arteries and themicrocirculation, and autoimmune changes.

The type of systemic scleroderma known as CREST is not characterized byany skin tightening. CREST is characterized by Calcinosis (calciumdeposits), usually in the fingers; Raynaud's; loss of muscle control ofthe Esophagus, which can cause difficulty swallowing; Sclerodactyly, atapering deformity of the bones of the fingers; and Telangiectasia,small red spots on the skin of the fingers, face, or inside of themouth. Typically two of these symptoms is sufficient for diagnosis ofCREST. CREST may occur alone, or in combination with any other form ofScleroderma or with other autoimmune diseases.

Limited Scleroderma is characterized by tight skin limited to thefingers, along with either pitting digital ulcers (secondary toRaynaud's) and/or lung fibrosis. The skin of the face and neck may alsobe involved in limited scleroderma.

Diffuse Scleroderma is diagnosed whenever there is proximal tight skin.Proximal means located closest to the reference point. Proximal tightskin can be skin tightness above the wrists or above the elbows.Typically, a patient with skin tightness only between their elbows andtheir wrists will receive a diagnosis of either diffuse or limitedsystemic Scleroderma, depending on which meaning of proximal thediagnosing clinician uses.

The current therpaies for scleroderma include extracorporealphotophoresis following 6-methoxypsoralen, and autologous stem celltransplant,

The current treatments for scleroderma include the administration of thefollowing agents, penicillamine, cholchicine, interferon alpha,interpheron gamma, chlorambucil, cyclosporine, 5-fluorouracil,cyclophosphamide, minocycline, thalidomide, etanercept, or methotrexate.

In certain embodiments of the methods of invention, a patient can betreated with an anti-CD19 antibody prior, concurrent, or subsequent toany of the therapies disclosed above that are used for the treatment ofautoimmune diabetes. Moreover, anti-CD19 antibodies of the presentinvention may be administered in combination with any of the agentsnoted above.

5.25. Determining CD19 Density in a Sample or Subject

While not required, assays for CD19 density can be employed to furthercharacterize the patient's diagnosis. Methods of determining the densityof antibody binding to cells are known to those skilled in the art (See,e.g., Sato et al., J. Immunology 165:6635-6643 (2000); which discloses amethod of assessing cell surface density of specific CD antigens). Otherstandard methods include Scatchard analysis. For example, the antibodyor fragment can be isolated, radiolabeled, and the specific activity ofthe radiolabeled antibody determined. The antibody is then contactedwith a target cell expressing CD19. The radioactivity associated withthe cell can be measured and, based on the specific activity, the amountof antibody or antibody fragment bound to the cell determined.

Fluorescence activated flow cytometry can also be employed. Generally,the antibody or antibody fragment is bound to a target cell expressingCD19. A second reagent that binds to the antibody is then added, forexample, a flourochrome labeled anti-immunoglobulin antibody.Flourochrome staining can then be measured and used to determine thedensity of antibody or antibody fragment binding to the cell.

As another suitable method, the antibody or antibody fragment can bedirectly labeled with a detectable label, such as a fluorophore, andbound to a target cell. The ratio of label to protein is determined andcompared with standard beads with known amounts of label bound thereto.Comparison of the amount of label bound to the cell with the knownstandards can be used to calculate the amount of antibody bound to thecell.

In yet another aspect, the present invention provides a method fordetecting in vitro or in vivo the presence and/or density of CD19 in asample or individual. This can also be useful for monitoring disease andeffect of treatment and for determining and adjusting the dose of theantibody to be administered. The in vivo method can be performed usingimaging techniques such as PET (positron emission tomography) or SPECT(single photon emission computed tomography). One could also label ananti-CD19 antibody with Indium using a covalently attached chelator. Theresulting antibody can be imaged using standard gamma cameras the sameway as ZEVALIN™ (ipritumomab tiuxetan) (Indium labeled anti-CD20 mAb)(Biogen Idec, Cambridge Mass.) is used to image CD20 antigen.

In one embodiment, the in vivo method can be performed by contacting asample to be tested, optionally along with a control sample, with ahuman anti-CD19 antibody under conditions that allow for formation of acomplex between an antibody of the invention and the human CD19 antigen.Complex formation is then detected (e.g., using fluorescent activatedflow cytometry or Western blotting). When using a control sample alongwith the test sample, a complex is detected in both samples and anystatistically significant difference in the formation of complexesbetween the samples is indicative of the presence of human CD19 in thetest sample.

In other embodiments, mean florescence intensity can be used as ameasure of CD19 density. In such embodiments, B cells are removed from apatient and stained with CD19 antibodies that have been labeled with aflorescent label and the fluorescence intensity is measured using flowcytometry. Fluorescence intensities can be measured and expressed as anaverage of intensity per B cell. Using such methods, mean florescenceintensities that are representative of CD19 density can be compared fora patient before and after treatment using methods and compositions ofthe invention, or between patients and normal levels of hCD19 on Bcells.

In patients where the density of CD19 expression on B cells has beendetermined, the density of CD19 may influence the determination and/oradjustment of the dosage and/or treatment regimen used with an anti-CD19antibody of compositions and methods of the invention. For example,where density of CD19 is high, it may be possible to use anti-CD19antibodies that less efficiently mediate ADCC in humans. In certainembodiments, where the patient treated using compositions and methods ofthe invention has a low CD19 density, a higher dosage of an anti-CD19antibody of compositions and methods of the invention may be used. Inother embodiments, where the patient treated using compositions andmethods of the invention has a low CD19 density, a low dosage of ananti-CD19 antibody of compositions and methods of the invention may beused. In certain embodiments, where the patient treated usingcompositions and methods of the invention has a high CD19 density, alower dosage of an anti-CD19 antibody of compositions and methods of theinvention may be used. In certain embodiments, CD19 density can becompared to CD20 density in a patient, CD19 density can be compared toan average CD19 density for humans or for a particular patientpopulation, or CD19 density can be compared to CD19 levels in thepatient prior to therapy or prior to onset of a B cell disease ordisorder. In certain embodiments, the patient treated using compositionsand methods of the invention has a B cell malignancy where CD19 ispresent on the surface of B cells.

5.26. Immunotherapeutic Protocols

Anti-CD19 antibody compositions used in the therapeuticregimen/protocols, referred to herein as “anti-CD19 immunotherapy” canbe naked antibodies, immunoconjugates and/or fusion proteins.Compositions of the invention can be used as a single agent therapy orin combination with other therapeutic agents or regimens. Anti-CD19antibodies or immunoconjugates can be administered prior to,concurrently with, or following the administration of one or moretherapeutic agents. Therapeutic agents that can be used in combinationtherapeutic regimens with compositions of the invention include anysubstance that inhibits or prevents the function of cells and/or causesdestruction of cells. Examples include, but are not limited to,radioactive isotopes, chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

The therapeutic regimens described herein, or any desired treatmentregimen can be tested for efficacy using a transgenic animal model whichexpresses human CD19 antigen in place of native CD19 antigen. Thus, ananti-CD19 antibody treatment regimen can be tested in an animal model todetermine efficacy before administration to a human.

Anti-CD19 antibodies, compositions and methods may be practiced to treatB cell diseases, including B cell malignancies. The term “B cellmalignancy” includes any malignancy that is derived from a cell of the Bcell lineage. Exemplary B cell malignancies include, but are not limitedto: B cell subtype non-Hodgkin's lymphoma (NHL) including lowgrade/follicular, NHL, small lymphocytic (SL) NHL, intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL; mantle-cell lymphoma, and bulky disease NHL;Burkitt's lymphoma; multiple myeloma; pre-B acute lymphoblastic leukemiaand other malignancies that derive from early B cell precursors; commonacute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL)including including immunoglobulin-mutated CLL andimmunoglobulin-unmutated CLL; hairy cell leukemia; Null-acutelymphoblastic leukemia; Waldenstrom's Macroglobulinemia; diffuse large Bcell lymphoma (DLBCL) including germinal center B cell-like (GCB) DLBCL,activated B cell-like (ABC) DLBCL, and type 3 DLBCL; pro-lymphocyticleukemia; light chain disease; plasmacytoma; osteosclerotic myeloma;plasma cell leukemia; monoclonal gammopathy of undetermined significance(MGUS); smoldering multiple myeloma (SMM); indolent multiple myeloma(IMM); Hodgkin's lymphoma including classical and nodular lymphocytepre-dominant type; lymphoplasmacytic lymphoma (LPL); and marginal-zonelymphoma including gastric mucosal-associated lymphoid tissue (MALT)lymphoma.

In a further embodiment the invention can be employed to treat mature Bcell malignancies (i.e., express Ig on the cell surface) including butnot limited to follicular lymphoma, mantle-cell lymphoma, Burkitt'slymphoma, multiple myeloma, diffuse large B-cell lymphoma (DLBCL)including germinal center B cell-like (GCB) DLBCL, activated B cell-like(ABC) DLBCL, and type 3 DLBCL, Hodgkin's lymphoma including classicaland nodular lymphocyte pre-dominant type, lymphoplasmacytic lymphoma(LPL), marginal-zone lymphoma including gastric mucosal-associatedlymphoid tissue (MALT) lymphoma, and chronic lymphocytic leukemia (CLL)including immunoglobulin-mutated CLL and immunoglobulin-unmutated CLL.

Further, CD19 is expressed earlier in B cell development than, forexample, CD20, and is therefore particularly suited for treating pre-Bcell and immature B cell malignancies (i.e., do not express Ig on thecell surface), for example, in the bone marrow. Illustrative pre-B celland immature B cell malignancies include but are not limited to acutelymphoblastic leukemia

In other particular embodiments, the invention can be practiced to treatextranodal tumors.

5.27. Anti-CD19 Immunotherapy

In accordance with the present invention “anti-CD19 immunotherapy”encompasses the administration of any of the anti-CD19 antibodies of theinvention in accordance with any therapeutic regimen described herein.Anti-CD19 antibodies can be administered as naked antibodies, orimmunoconjugates or fusion proteins.

Anti-CD19 immunotherapy encompasses the administration of an anti-CD19antibody as a single agent therapeutic for the treatment of a B cellmalignancy. Anti-CD19 immunotherapy encompasses methods of treating anearly stage disease resulting from a B cell malignancy. Anti-CD19immunotherapy encompasses methods of treating a B cell malignancywherein an anti-CD19 antibody mediates ADCC. Anti-CD19 immunotherapyencompasses methods of treating a B cell malignancy wherein an anti-CD19antibody is administered before the patient has received any treatmentfor the malignancy, whether that therapy is chemotherapy, radio chemicalbased therapy or surgical therapy.

In one embodiment, a human subject having a B cell malignancy can betreated by administering a human or humanized antibody that may be ableto mediate human ADCC. In cases of early stage disease, or single agenttherapies, any anti-CD19 antibody that may mediate ADCC can be used inthe human subjects (including murine and chimeric antibodies); however,human and humanized antibodies may be preferred.

Antibodies of IgG1 or IgG3 human isotypes are in some cases preferredfor therapy. However, the IgG2 or IgG4 human isotypes can be used aswell, provided they have the relevant effector function, for examplehuman ADCC. Such effector function can be assessed by measuring theability of the antibody in question to mediate target cell lysis byeffector cells in vitro or in vivo.

In one embodiment, the dose of antibody used should be sufficient todeplete circulating B cells. Progress of the therapy can be monitored inthe patient by analyzing blood samples. Other signs of clinicalimprovement can be used to monitor therapy.

Methods for measuring depletion of B cells that can be used inconnection with compositions and methods of the invention are well knownin the art and include, but are not limited to the followingembodiments. In one embodiment, circulating B cells depletion can bemeasured with flow cytometry using a reagent other than an anti-CD19antibody that binds to B cells to define the amount of B cells. In otherembodiments, B cell levels in the blood can be monitored using standardserum analysis. In such embodiments, B cell depletion is indirectlymeasured by defining the amount to an antibody known to be produced by Bcells. The level of that antibody is then monitored to determine thedepletion and/or functional depletion of B cells. In another embodiment,B cell depletion can be measured by immunochemical staining to identifyB cells. In such embodiments, B cells or tissues or serum comprising Bcells extracted from a patient can be placed on microscope slides,labeled and examined for presence or absence. In related embodiments, acomparison is made between B cells extracted prior to therapy and aftertherapy to determine differences in the presence of B cells.

Tumor burden can be measured and used in connection with compositionsand methods of the invention. Methods for measuring tumor burden arewell known in the art and include, but are not limited to the followingembodiments. In certain embodiments, PET scans can be used to measuremetabolic activity and identify areas of higher activity which areindicative of tumors. CT scans and MRI can also be used to examine softtissue for the presence and size of tumors. In other embodiments, bonescan can be used to measure tumor volume and location. In yet otherembodiments, tumor burden can be measured by examining the blood flowinto and out of a tumor using doppler technology (e.g., ultrasound). Insuch embodiments, changes in blood flow over time or deviations fromnormal blood flow in the appropriate tissue of a patient can be used tocalculate an estimate to tumor burden. Such methods for measuring tumorburden can be used prior to and following methods of treatment of theinvention.

In certain embodiments of methods of the invention B cells are depletedand/or tumor burden is decreased while ADCC function is maintained.

In embodiments of the invention where an anti-CD19 antibody isadministered as a single agent therapy, the invention contemplates useof different treatment regimens.

According to certain aspects of the invention, an anti-CD19 antibodyused in compositions and methods of the invention, is a naked antibody.In related embodiments, the dose of naked anti-CD19 antibody used is atleast about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5 mg/kg of body weight of a patient. In certainembodiments, the dose of naked anti-CD19 antibody used is at least about1 to 10, 5 to 15, 10 to 20, or 15 to 25 mg/kg of body weight of apatient. In certain embodiments, the dose of naked anti-CD19 antibodyused is at least about 1 to 20, 3 to 15, or 5 to 10 mg/kg of body weightof a patient. In other embodiments, the dose of naked anti-CD19 antibodyused is at least about 5, 6, 7, 8, 9, or 10 mg/kg of body weight of apatient.

In certain embodiments, the dose comprises about 375 mg/m2 of anti-CD19antibody administered weekly for 4 to 8 consecutive weeks. In certainembodiments, the dose is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 mg/kg of body weight of the patient administeredweekly for 4 to 8 consecutive weeks.

The exemplary doses of anti-CD19 antibody described above can beadministered as described in Section 5.21.3. In one embodiment, theabove doses are single dose injections. In other embodiments, the dosesare administered over a period of time. In other embodiments, the dosesare administered multiple times over a period of time. The period oftime may be measured in days, weeks, or months. Multiple doses of ananti-CD19 antibody can be administered at intervals suitable to achievea therapeutic benefit while balancing toxic side effects. For example,where multiple doses are used, it may be preferred to time the intervalsto allow for recovery of the patient's monocyte count prior to therepeat treatment with antibody. This dosing regimen will optimize theefficiency of treatment, since the monocyte population reflects ADCCfunction in the patient.

In certain embodiments, compositions of the invention are administeredto a human patient as long as the patient is responsive to therapy. Inother embodiments, compositions of the invention are administered to ahuman patient as long as the patient's disease does not progress. Inrelated embodiments, compositions of the invention are administered to ahuman patient until a patient's disease does not progress or has notprogressed for a period of time, then the patient is not administeredcompositions of the invention unless the disease reoccurs or begins toprogress again. For example, a patient can be treated with any of theabove doses for about 4 to 8 weeks, during which time the patient ismonitored for disease progression. If disease progression stops orreverses, then he patient will not be administered compositions of theinvention until that patient relapses, i.e., the disease being treatedreoccurs or progresses. Upon this reoccurrence or progression, thepatient can be treated again with the same dosing regimen initially usedor using other doses described above.

In certain embodiments, compositions of the invention can beadministered as a loading dose followed by multiple lower doses(maintenance doses) over a period of time. In such embodiments, thedoses may be timed and the amount adjusted to maintain effective B celldepletion. In certain embodiments, the loading dose is about 10, 11, 12,13, 14, 15, 16, 17, or 18 mg/kg of patient body weight and themaintenance dose is at least about 5 to 10 mg/kg of patient body weight.In other embodiments, the maintenance dose is administered at intervalsof every 7, 10, 14 or 21 days. The maintenance doses can be continuedindefinitely, until toxicity is present, until platelet count decreases,until there is no disease progression, until the patient exhibitsimmunogenicity, or until disease progresses to a terminal state. In yetother embodiments, compositions of the invention are administered to ahuman patient until the disease progresses to a terminal stage.

In embodiments of the invention where circulating monocyte levels of apatient are monitored as part of a treatment regimen, doses of anti-CD19antibody administered may be spaced to allow for recovery of monocytecount. For example, a composition of the invention may be administeredat intervals of every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

In embodiments of the invention where an anti-CD19 antibody isconjugated to or administered in conjunction with a toxin, one skilledin the art will appreciate that the dose of anti-CD19 antibody can beadjusted based on the toxin dose and that the toxin dose will depend onthe specific type of toxin being used. Typically, where a toxin is used,the dose of anti-CD19 antibody will be less than the dose used with anaked anti-CD19 antibody. The appropriate dose can be determined for aparticular toxin using techniques well known in the art. For example, adose range study can be conducted to determine the maximum tolerateddose of anti-CD19 antibody when administered with or conjugated to atoxin.

In embodiments of the invention where an anti-CD19 antibody isconjugated to or administered in conjunction with a radiotherapeuticagent, the dose of the anti-CD19 antibody will vary depending on theradiotherapeutic used. In certain embodiments, a two step process isused. First, the human patient is administered a composition comprisinga naked anti-CD19 antibody and about 6, 7, 8, 9, or 10 days later asmall amount of the radiotherapeutic is administered. Second, once thetolerance, distribution, and clearance of the low dose therapy has beendetermined, the patient is administered a dose of the naked anti-CD19antibody followed by a therapeutic amount of the radiotherapeutic isadministered. Such treatment regimens are similar to those approved fortreatment of Non-Hodgkin's lymphoma using ZEVALIN™ (ipritumomabtiuxetan) (Indium labeled anti-CD20 mAb) (Biogen Idec) or BEXXAR™(tosiumomab) (GSK, Coulter Pharmaceutical).

5.28. Combination with Chemotherapeutic Agents

Anti-CD19 immunotherapy (using naked antibody, immunoconjugates, orfusion proteins) can be used in conjunction with other therapiesincluding but not limited to, chemotherapy, radioimmunotherapy (RIT),chemotherapy and external beam radiation (combined modality therapy,CMT), or combined modality radioimmunotherapy (CMRIT) alone or incombination, etc. In certain embodiments, an anti-CD19 antibody therapyof the present invention can be administered in conjunction with CHOP(Cyclophosphamide-Hydroxydoxorubicin-Oncovin(vincristine)-Prednisolone), the most common chemotherapy regimen fortreating non-Hodgkin's lymphoma. As used herein, the term “administeredin conjunction with” means that an anti-CD19 immunotherapy can beadministered before, during, or subsequent to the other therapyemployed.

In certain embodiments, an anti-CD19 immunotherapy is in conjunctionwith a cytotoxic radionuclide or radiotherapeutic isotope. For example,an alpha-emitting isotope such as ²²⁵Ac, ²²⁴Ac, ²¹¹At, ²¹²Bi, ²¹³Bi,²¹²Pb, ²²⁴Ra, or ²²³Ra. The cytotoxic radionuclide may also be abeta-emitting isotope such as ¹⁸⁶Rc, ¹⁸⁸Rc, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹⁷⁷Lu,¹⁵³Sm, ¹⁶⁶Ho or ⁶⁴Cu. Further, the cytotoxic radionuclide may emit Augerand low energy electrons and include the isotopes ¹²⁵I ¹²³I or ⁷⁷Br. Inother embodiments the isotope may be ¹⁹⁸Au, ³²P, and the like. Incertain embodiments, the amount of the radionuclide administered to thesubject is between about 0.001 mCi/kg and about 10 mCi/kg.

In some embodiments, the amount of the radionuclide administered to thesubject is between about 0.1 mCi/kg and about 1.0 mCi/kg. In otherembodiments, the amount of the radionuclide administered to the subjectis between about 0.005 mCi/kg and 0.1 mCi/kg.

In certain embodiments, an anti-CD19 immunotherapy is in conjunctionwith a chemical toxin or chemotherapeutic agent. The chemical toxin orchemotherapeutic agent may be selected from the group consisting of anenediyne such as calicheamicin and esperamicin; duocarmycin,methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine,mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.

Suitable chemical toxins or chemotherapeutic agents that can be used incombination therapies with an anti-CD19 immunotherapy include members ofthe enediyne family of molecules, such as calicheamicin and esperamicin.Chemical toxins can also be taken from the group consisting ofduocarmycin (see, e.g., U.S. Pat. No. 5,703,080 and U.S. Pat. No.4,923,990), methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C,vindesine, mitomycin C, cis-platinum, etoposide, bleomycin and5-fluorouracil. Examples of chemotherapeutic agents also includeAdriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see, U.S. Pat. No.4,675,187), Melphalan and other related nitrogen mustards.

In other embodiments, for example, “CVB” (1.5 g/m² cyclophosphamide,200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) can be used incombination therapies of the invention. CVB is a regimen used to treatnon-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51:18 (1993).Other suitable combination chemotherapeutic regimens are well-known tothose of skill in the art. See, for example, Freedman et al.,“Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition,Holland et al. (eds.), pp. 2028-2068 (Lea & Febiger 1993). As anillustration, first generation chemotherapeutic regimens for treatmentof intermediate-grade non-Hodgkin's lymphoma include C-MOPP(cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP(cyclophosphamide, doxorubicin, vincristine, and prednisone). A usefulsecond generation chemotherapeutic regimen is m-BACOD (methotrexate,bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate and brostatin-1. In a multimodal therapy, both chemotherapeuticdrugs and cytokines are co-administered with an antibody,immunoconjugate or fusion protein according to the present invention.The cytokines, chemotherapeutic drugs and antibody, immunoconjugate orfusion protein can be administered in any order, or together.

Other toxins that may be used in compositions and methods of theinvention include poisonous lectins, plant toxins such as ricin, abrin,modeccin, botulina and diphtheria toxins. Of course, combinations of thevarious toxins could also be coupled to one antibody molecule therebyaccommodating variable cytotoxicity. Illustrative of toxins which aresuitably employed in combination therapies of the invention are ricin,abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas exotoxin. See, for example, Pastan et al., Cell 47:641(1986), and Goldenberg et al., Cancer Journal for Clinicians 44:43(1994). Enzymatically active toxins and fragments thereof which can beused include diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

Suitable toxins and chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985). Other suitable toxins and/orchemotherapeutic agents are known to those of skill in the art.

An anti-CD19 immunotherapy of the present invention may also be inconjunction with a prodrug-activating enzyme which converts a prodrug(e.g., a peptidyl chemotherapeutic agent, see, WO81/01145) to an activeanti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278. The enzyme component of such combinations includes any enzymecapable of acting on a prodrug in such a way so as to covert it into itsmore active, cytotoxic form. The term “prodrug” as used in thisapplication refers to a precursor or derivative form of apharmaceutically active substance that is less cytotoxic to tumor cellscompared to the parent drug and is capable of being enzymaticallyactivated or converted into the more active parent form. See, e.g.,Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stellaet al., Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al. (ed.), pp. 247-267, HumanaPress (1985). Prodrugs that can be used in combination with anti-CD19antibodies include, but are not limited to, phosphate-containingprodrugs, thiophosphate-containing prodrugs, sulfate-containingprodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, a-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

In certain embodiments, administration of compositions and methods ofthe invention may enable the postponement of toxic therapy and may helpavoid unnecessary side effects and the risks of complications associatedwith chemotherapy and delay development of resistance to chemotherapy.In certain embodiments, toxic therapies and/or resistance to toxictherapies is delayed in patients administered compositions and methodsof the invention delay for up to about 6 months, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 years.

5.29. Combination with Therapeutic Antibodies

An anti-CD19 immunotherapy described herein may be administered incombination with other antibodies, including, but not limited to,anti-CD20 mAb, anti-CD52 mAb, anti-CD22 antibody, and anti-CD20antibodies, such as RITUXAN™ (C2B8; RITUXIMAB™; IDEC Pharmaceuticals).Other examples of therapeutic antibodies that can be used in combinationwith antibodies of the invention or used in compositions of theinvention include, but are not limited to, HERCEPTIN™ (Trastuzumab;Genentech), MYLOTARG™ (Gemtuzumab ozogamicin; Wyeth Pharmaceuticals),CAMPATH™ (Alemtuzumab; Berlex), ZEVALIN™ (Ipritumomab tiuxetan; BiogenIdec), BEXXAR™ (Tositumomab; GlaxoSmithKline Corixa), ERBITUX™(Cetuximab; Imclone), and AVASTIN™ (Bevacizumab; Genentech).

An anti-CD19 immunotherapy described herein may be administered incombination with an antibody specific for an Fc receptor selected fromthe group consisting of FcγRI, FcγRIIA, FcγRIIB, FcγRIII and/or FcγRIV.In a specific embodiment, an anti-CD19 immunotherapy described hereinmay be administered in combination with an antibody specific for FcγRIIBAnti-FcγRIIB antibodies suitable for this purpose have been described inUS Patent Application Publication No. 2004185045 (U.S. Pat. No.7,425,620), PCT Publication Nos. WO05051999A, WO05018669 and WO04016750.

In certain embodiments, an anti-CD19 and an anti-CD20 and/or ananti-CD22 mAb and/or an anti-CD52 mAb can be administered, optionally inthe same pharmaceutical composition, in any suitable ratio. Toillustrate, the ratio of the anti-CD19 and anti-CD20 antibody can be aratio of about 1000:1, 500:1, 250:1, 100:1, 90:1, 80:1, 70:1, 60;1,50:1, 40:1, 30:1. 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1,11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90. 1:100,1:250, 1:500 or 1:1000 or more. Likewise, the ratio of the anti-CD19 andanti-CD22 antibody can be a ratio of about 1000:1, 500:1, 250:1, 100:1,90:1, 80:1, 70:1, 60;1, 50:1, 40:1, 30:1. 20:1, 19:1, 18:1, 17:1, 16:1,15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1,2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60,1:70, 1:80, 1:90. 1:100, 1:250, 1:500 or 1:1000 or more. Similarly, theratio of the anti-CD19 and anti-CD52 antibody can be a ratio of about1000:1, 500:1, 250:1, 100:1, 90:1, 80:1, 70:1, 60;1, 50:1, 40:1, 30:1.20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19,1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90. 1:100, 1:250, 1:500 or1:1000 or more.

5.30. Combination Compounds that Enhance Monocyte or Macrophage Function

In certain embodiments of methods of the invention, a compound thatenhances monocyte or macrophage function (e.g., at least about 25%, 50%,75%, 85%, 90%, 95% or more) can be used in conjunction with an anti-CD19immunotherapy. Such compounds are known in the art and include, withoutlimitation, cytokines such as interleukins (e.g., IL-12), andinterferons (e.g., alpha or gamma interferon).

The compound that enhances monocyte or macrophage function orenhancement can be formulated in the same pharmaceutical composition asthe antibody, immunoconjugate or antigen-binding fragment. Whenadministered separately, the antibody/fragment and the compound can beadministered concurrently (within a period of hours of each other), canbe administered during the same course of therapy, or can beadministered sequentially (i.e., the patient first receives a course ofthe antibody/fragment treatment and then a course of the compound thatenhances macrophage/monocyte function or vice versa). In suchembodiments, the compound that enhances monocyte or macrophage functionis administered to the human subject prior to, concurrently with, orfollowing treatment with other therapeutic regimens and/or compositionsof the invention. In one embodiment, the human subject has a bloodleukocyte, monocyte, neutrophil, lymphocyte, and/or basophil count thatis within the normal range for humans. Normal ranges for human bloodleukocytes (total) is about 3.5-about 10.5 (10⁹/L). Normal ranges forhuman blood neutrophils is about 1.7-about 7.0 (10⁹/L), monocytes isabout 0.3-about 0.9 (10⁹/L), lymphocytes is about 0.9-about 2.9 (10⁹/L),basophils is about 0-about 0.3 (10⁹/L), and eosinophils is about0.05-about 0.5 (10⁹/L). In other embodiments, the human subject has ablood leukocyte count that is less than the normal range for humans, forexample at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or0.8 (10⁹/L) leukocytes.

This embodiment of the invention can be practiced with the antibodies,immunocongugates or antibody fragments of the invention or with otherantibodies known in the art and is particularly suitable for subjectsthat are resistant to anti-CD22, anti-CD52 and/or anti-CD20 antibodytherapy (for example, therapy with existing antibodies such as C2B8),subjects that are currently being or have previously been treated withchemotherapy, subjects that have had a relapse in a B cell disorder,subjects that are immunocompromised, or subjects that otherwise have animpairment in macrophage or monocyte function. The prevalence ofpatients that are resistant to therapy or have a relapse in a B celldisorder may be attributable, at least in part, to an impairment inmacrophage or monocyte function. Thus, the invention provides methods ofenhancing ADCC and/or macrophage and/or monocyte function to be used inconjunction with the methods of administering anti-CD19 antibodies andantigen-binding fragments.

5.31. Combination with Immunoregulatory Agents

An anti-CD19 immunotherapy of the invention may also be in conjunctionwith an immunoregulatory agent. In this approach, a chimeric, human orhumanized anti-CD19 antibody can be used. The term “immunoregulatoryagent” as used herein for combination therapy refers to substances thatact to suppress, mask, or enhance the immune system of the host. Thiswould include substances that suppress cytokine production, downregulateor suppress self-antigen expression, or mask the MHC antigens. Examplesof such agents include 2-amino-6-aryl-5-substituted pyrimidines (see,U.S. Pat. No. 4,665,077), azathioprine (or cyclophosphamide, if there isan adverse reaction to azathioprine); bromocryptine; glutaraldehyde(which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone,methylprednisolone, and dexamethasone; cytokine or cytokine receptorantagonists including anti-interferon-γ, -β, or -α antibodies;anti-tumor necrosis factor-α antibodies; anti-tumor necrosis factor-βantibodies; anti-interleukin-2 antibodies and anti-IL-2 receptorantibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin;pan-T antibodies, for example anti-CD3 or anti-CD4/CD4a antibodies;soluble peptide containing a LFA-3 binding domain (WO 90/08187 publishedJul. 26, 1990); streptokinase; TGF-β; streptodomase; RNA or DNA from thehost; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (U.S.Pat. No. 5,114,721); T-cell receptor fragments (Offner et al., Science251:430-432 (1991); WO 90/11294; and WO 91/01133); and T-cell receptorantibodies (EP 340,109) such as T10B9. Examples of cytokines include,but are not limited to lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-a;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoiotin (TPO); nerve growth factors such as NGF-α;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-α; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CgP (GM-CSP); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2,1L-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-1 I, IL-12, IL-15; a tumornecrosis factor such as TNF-α or TNF-β; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines. Incertain embodiments, the methods further include administering to thesubject one or more immunomodulatory agents, for example a cytokine.Suitable cytokines may be selected from the group consisting ofinterleukin-1 (IL-1), IL-2, IL-3, IL-12, IL-15, IL-18, G-CSF, GM-CSF,thrombopoietin, and γ interferon.

These immunoregulatory agents are administered at the same time or atseparate times from anti-CD19 antibodies. The preferred immunoregulatoryagent will depend on many factors, including the type of disorder beingtreated, as well as the patient's history, but the agent frequently maybe selected from cyclosporin A, a glucocorticostcroid (for exampleprednisone or methylprednisolone), OKT-3 monoclonal antibody,azathioprine, bromocryptine, heterologous anti-lymphocyte globulin, or amixture thereof.

5.32. Combination with Other Therapeutic Agents

Agents that act on the tumor neovasculature can also be used inconjunction with anti-CD19 immunotherapy and include tubulin-bindingagents such as combrestatin A4 (Griggs et al., Lancet Oncol. 2:82,(2001)) and angiostatin and endostatin (reviewed in Rosen, Oncologist5:20 (2000), incorporated by reference herein). Immunomodulatorssuitable for use in combination with anti-CD19 antibodies include, butare not limited to, of α-interferon, γ-interferon, and tumor necrosisfactor alpha (TNFα). In certain embodiments, the therapeutic agents usedin combination therapies using compositions and methods of the inventionare peptides.

In certain embodiments, an anti-CD19 immunotherapy is in conjunctionwith one or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. Structural analogues of calicheamicinwhich may be used include, but are not limited to, γ1^(I), γ2^(I),γ3^(I),N-acetyl-γ1^(I), PSAG and 011 Hinman et al., Cancer Research53:3336-3342 (1993) and Lode et al., Cancer Research 58: 2925-2928(1998)).

A fusion protein comprising an anti-CD19 antibody and a cytotoxic agentmay also be made, e.g., by recombinant techniques or peptide synthesis.γ

In yet another embodiment, an anti-CD19 antibody may be conjugated to a“receptor” (such as streptavidin) for utilization in tumor pretargetingwherein the antagonist-receptor conjugate is administered to thepatient, followed by removal of unbound conjugate from the circulationusing a clearing agent and then administration of a “ligand” (e.g.,avidin) which is conjugated to a therapeutic agent (e.g., aradionucleotide).

In certain embodiments, a treatment regimen includes compounds thatmitigate the cytotoxic effects of an anti-CD19 antibody composition.Such compounds include analgesics (e.g., acetaminophen),bisphosphonates, antihistamines (e.g., chlorpheniramine maleate), andsteroids (e.g., dexamethasone, retinoids, deltoids, betamethasone,cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids,mineralocorticoids, estrogen, testosterone, progestins).

In certain embodiments, the therapeutic agent used in combination withan anti-CD19 immunotherapy is a small molecule (i.e., inorganic ororganic compounds having a molecular weight of less than about 2500daltons). For example, libraries of small molecules may be commerciallyobtained from Specs and BioSpecs B.V. (Rijswijk, The Netherlands),Chembridge Corporation (San Diego, Calif.), Comgenex USA Inc.(Princeton, N.J.), and Maybridge Chemicals Ltd. (Cornwall PL34 OHW,United Kingdom).

In certain embodiments an anti-CD19 immunotherapy can be administered incombination with an anti-bacterial agent. Non-limiting examples ofanti-bacterial agents include proteins, polypeptides, peptides, fusionproteins, antibodies, nucleic acid molecules, organic molecules,inorganic molecules, and small molecules that inhibit and/or reduce abacterial infection, inhibit and/or reduce the replication of bacteria,or inhibit and/or reduce the spread of bacteria to other cells orsubjects. Specific examples of anti-bacterial agents include, but arenot limited to, antibiotics such as penicillin, cephalosporin, imipenem,axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol,erythromycin, clindamycin, tetracycline, streptomycin, tobramycin,gentamicin, amikacin, kanamycin, neomycin, spectinomycin, trimethoprim,norfloxacin, rifampin, polymyxin, amphotericin B, nystatin,ketocanazole, isoniazid, metronidazole, and pentamidine.

In certain embodiments an anti-CD19 immunotherapy can be administered incombination with an anti-fungal agent. Specific examples of anti-fungalagents include, but are not limited to, azole drugs (e.g., miconazole,ketoconazole (NIZORAL®), caspofungin acetate (CANCIDAS®), imidazole,triazoles (e.g., fluconazole (DIFLUCAN®)), and itraconazole (SPORANOX°),polyene (e.g., nystatin, amphotericin B (FUNGIZONE®), amphotcricin Blipid complex (“ABLC”) (ABELCET®), amphotcricin B colloidal dispersion(“ABCD”) (AMPHOTEC®), liposomal amphotericin B (AMB1SONE®)), potassiumiodide (KI), pyrimidine (e.g., flucytosine (ANCOBON®), and voriconazole(VFEND®)). Administration of anti bacterial and anti-fungal agents canmitigate the effects or escalation of infectious disease that may occurin methods of the invention where a patient's B cells are significantlydepleted.

In certain embodiments of the invention, an anti-CD19 immunotherapy canbe administered in combination with one or more of the agents describedabove to mitigate the toxic side effects that may accompanyadministration of compositions of the invention. In other embodiments,an anti-CD19 immunotherapy can be administered in combination with oneor more agents that are well known in the art for use in mitigating theside effects of antibody administration, chemotherapy, toxins, or drugs.

In certain embodiments of the invention where an anti-CD19 immunotherapyis administered to treat multiple myeloma, compositions of the inventionmay be administered in combination with or in treatment regimens withhigh-dose chemotherapy (melphalan, melphalan/prednisone (MP),vincristine/doxorubicin/dexamethasone (VAD), liposomaldoxorubicin/vincristine, dexamethasone (DVd), cyclophosphamide,etoposide/dexamethasone/cytarabine, cisplatin (EDAP)), stem celltransplants (e.g., autologous stem cell transplantation or allogeneicstem cell transplantation, and/or mini-allogeneic (non-myeloablative)stem cell transplantation), radiation therapy, steroids (e.g.,corticosteroids, dexamethasone, thalidomide/dexamethasone, prednisone,melphalan/prednisone), supportive therapy (e.g., bisphosphonates, growthfactors, antibiotics, intravenous immunoglobulin, low-dose radiotherapy,and/or orthopedic interventions), THALOMID™ (thalidomide, Celgene),and/or VELCADE™ (bortezomib, Millennium).

In embodiments of the invention where an anti-CD19 immunotherapy isadministered in combination with another antibody or antibodies and/oragent, the additional antibody or antibodies and/or agents can beadministered in any sequence relative to the administration of theantibody of this invention. For example, the additional antibody orantibodies can be administered before, concurrently with, and/orsubsequent to administration of an anti-CD19 antibody or immunoconjugateto the human subject. The additional antibody or antibodies can bepresent in the same pharmaceutical composition as an antibody of theinvention, and/or present in a different pharmaceutical composition. Thedose and mode of administration of an antibody of this invention and thedose of the additional antibody or antibodies can be the same ordifferent, in accordance with any of the teachings of dosage amounts andmodes of administration as provided in this application and as are wellknown in the art.

5.33. Use of Anti-CD19 Antibodies in Diagnosing B Cell Malignancies

The present invention also encompasses anti-CD19 antibodies, andcompositions thereof, that immunospecifically bind to the human CD19antigen, which anti-CD19 antibodies are conjugated to a diagnostic ordetectable agent. In certain embodiments, the antibodies are human orhumanized anti-CD19 antibodies. Such anti-CD19 antibodies can be usefulfor monitoring or prognosing the development or progression of a B cellmalignancy as part of a clinical testing procedure, such as determiningthe efficacy of a particular therapy. Such diagnosis and detection canbe accomplished by coupling an anti-CD19 antibody thatimmunospecifically binds to the human CD19 antigen to a detectablesubstance including, but not limited to, various enzymes, such as butnot limited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, such asbut not limited to, streptavidinlbiotin and avidin/biotin; fluorescentmaterials, such as but not limited to, umbelliferone, fluorescein,fluorescein isothiocynate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; luminescent materials,such as but not limited to, luminol; bioluminescent materials, such asbut not limited to, luciferase, luciferin, and aequorin; radioactivematerials, such as but not limited to iodine (¹³¹I, ¹²⁵I, ¹²¹I, ¹²¹I)carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In,¹¹¹In,), and technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶H0, 90-y, ⁴⁷Se, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Cn, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tinpositron emitting metals using various positron emission tomographies,noradioactive paramagnetic metal ions, and molecules that areradiolabelled or conjugated to specific radioisotopes. Any detectablelabel that can be readily measured can be conjugated to an anti-CD19antibody and used in diagnosing B cell malignancies. The detectablesubstance may be coupled or conjugated either directly to an antibody orindirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, e.g., U.S.Pat. No. 4,741,900 for metal ions which can be conjugated to antibodiesfor use as a diagnostics according to the present invention. In certainembodiments, the invention provides for diagnostic kits comprising ananti-CD19 antibody conjugated to a diagnostic or detectable agent.

5.34. Use of Anti-CD19 Antibodies in Monitoring Immune Reconstitution

The present invention also encompasses anti-CD19 antibodies, andcompositions thereof, that immunospecifically bind to the human CD19antigen, which anti-CD19 antibodies are conjugated to a diagnostic ordetectable agent. In certain embodiments, the antibodies are human orhumanized anti-CD19 antibodies. Such anti-CD19antibodies can be usefulfor monitoring immune system reconstitution following immunosuppressivetherapy or bone marrow transplantation. Such monitoring can beaccomplished by coupling an anti-CD19 antibody that immunospecificallybinds to the human CD19 antigen to a detectable substance including, butnot limited to, various enzymes, such as, but not limited to,horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidinlbiotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as, but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I) carbon(¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), and technetium(⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru,⁶⁸Ge, ⁵⁷Co ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron-emitting metals using various positron-emissiontomographies, noradioactive paramagnetic metal ions, and molecules thatare radiolabelled or conjugated to specific radioisotopes. Anydetectable label that can be readily measured can be conjugated to ananti-CD19 antibody and used in diagnosing an autoimmune disease ordisorder. The detectable substance may be coupled or conjugated eitherdirectly to an antibody or indirectly, through an intermediate (such as,for example, a linker known in the art) using techniques known in theart. See, e.g., U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as a diagnostics according to thepresent invention. In certain embodiments, the invention provides fordiagnostic kits comprising an anti-CD19 antibody conjugated to adiagnostic or detectable agent.

5.35. Use of Anti-CD19 Antibodies in Diagnosing Autoimmune Diseases orDisorders

The present invention also encompasses anti-CD19 antibodies, andcompositions thereof, that immunospecifically bind to the human CD19antigen, which anti-CD19 antibodies are conjugated to a diagnostic ordetectable agent. In certain embodiments, the antibodies are human orhumanized anti-CD19 antibodies. Such anti-CD19 antibodies can be usefulfor monitoring or prognosing the development or progression of anautoimmune disease or disorder as part of a clinical testing procedure,such as determining the efficacy of a particular therapy. Such diagnosisand detection can be accomplished by coupling an anti-CD19 antibody thatimmunospecifically binds to the human CD19 antigen to a detectablesubstance including, but not limited to, various enzymes, such as butnot limited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, such asbut not limited to, streptavidinlbiotin and avidin/biotin; fluorescentmaterials, such as but not limited to, umbelliferone, fluorescein,fluorescein isothiocynate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; luminescent materials,such as but not limited to, luminol; bioluminescent materials, such asbut not limited to, luciferase, luciferin, and aequorin; radioactivematerials, such as but not limited to iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I),carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In,¹¹¹In) and technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb,⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin; positron emitting metals usingvarious positron emission tomographies, noradioactive paramagnetic metalions, and molecules that are radiolabelled or conjugated to specificradioisotopes. Any detectable label that can be readily measured can beconjugated to an anti-CD19 antibody and used in diagnosing an autoimmunedisease or disorder. The detectable substance may be coupled orconjugated either directly to an antibody or indirectly, through anintermediate (such as, for example, a linker known in the art) usingtechniques known in the art. See, e.g., U.S. Pat. No. 4,741,900 formetal ions which can be conjugated to antibodies for use as adiagnostics according to the present invention. In certain embodiments,the invention provides for diagnostic kits comprising an anti-CD19antibody conjugated to a diagnostic or detectable agent.

5.36. Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with a composition of the invention for theprevention, treatment, management or amelioration of a B cellmalignancy, or one or more symptoms thereof, potentiated by orpotentiating a B cell malignancy.

The present invention provides kits that can be used in theabove-described methods. In one embodiment, a kit comprises acomposition of the invention, in one or more containers. In anotherembodiment, a kit comprises a composition of the invention, in one ormore containers, and one or more other prophylactic or therapeuticagents useful for the prevention, management or treatment of a B cellmalignancy, or one or more symptoms thereof, potentiated by orpotentiating a B cell malignancy in one or more other containers. Thekit may further comprise instructions for preventing, treating, managingor ameliorating a B cell malignancy, as well as side effects and dosageinformation for method of administration. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

6. SPECIFIC EMBODIMENTS

1. A chimeric, humanized, or human monoclonal antibody or fragmentthereof that binds a human CD19 antigen.

2. The antibody of embodiment 1 comprising at least one CDR comprisingan amino acid sequence selected from the group consisting of: SEQ ID NO:6, 8, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, and 127.

3. The antibody of embodiment 1 comprising at least one framework regionof the HB12B-(A10-Jk4), HB12B-3649, or HB12B-364987 light chain variableregions.

4. The antibody of embodiment 1 comprising at least one framework regionof the HB12B-(3-72\JH4) or HB12B-9m heavy chain variable regions.

5. The antibody of embodiment 1 comprising at least one heavy chainpolypeptide comprising the amino acid sequence selected from the groupconsisting of: SEQ ID NO: 2, 18, 34, 44, 102, 103, 104, 105, 106, 107,108, and 109.

6. The antibody of embodiment 1 comprising at least one light chainpolypeptide comprising an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 4, 20, 52, 62, 68, 70, 110, 111, 112, and 113.

7. The antibody of embodiment 5 further comprising at least one lightchain CDR comprising the amino acid sequence selected from the groupconsisting of: SEQ ID NO: 12, 14, 16, 28, 30, 32, 123, 124, 125, 126,and 127.

8. The antibody of embodiment 3 further comprising at least one heavychain CDR comprising the amino acid sequence selected from the groupconsisting of: SEQ ID NO: 6, 8, 10, 22, 24, 26, 114, 115, 116, 117, 118,119, 120, 121, and 122.

9. The antibody of embodiment 1 comprising at least one heavy chainpolypeptide comprising the amino acid sequence selected from the groupconsisting of: SEQ ID NO: 2, 18, 34, 44, 102, 103, 104, 105, 106, 107,108, and 109, and at least one light chain polypeptide comprising anamino acid sequence selected from the group consisting of: SEQ ID NO: 4,20, 52, 62, 68, 70, 110, 111, 112, and 113.

10. The antibody of embodiment 1 comprising at least one light chainpolypeptide and at least one heavy chain polypeptide wherein said lightchain polypeptide comprises an amino acid sequence selected from thegroup consisting of: HB12A VK (SEQ ID NO: 4); and HB12B VK (SEQ IDNO:20), and wherein said heavy chain polypeptide comprises an amino acidsequence selected from the group consisting of: HB12A VH (SEQ ID NO: 2);and HB12B VH (SEQ ID NO:18).

11. The antibody of embodiment 1 comprising the HB12B-(3-72\JH4) heavychain variable region and the HB12B-3649 light chain variable region.

12. The antibody of embodiment 1 comprising a VH and a VK, wherein saidVH comprises the amino acid sequence of SEQ ID NO: 106, and wherein saidVK comprises the amino acid sequence of SEQ ID NO: 111.

13. A nucleic acid encoding a polypeptide comprising an amino acidsequence selected from the group consisting of: SEQ ID NO: 2, 18, 34,36, 38, 40, 42, 44, 102, 103, 104, 105, 106, 107, 108, and 109.

14. A nucleic acid encoding a polypeptide comprising an amino acidsequence selected from the group consisting of: SEQ ID NO: 4, 20, 52,62, 64, 66, 68, 70, 110, 111, 112, and 113.

15. A vector comprising the nucleic acid of embodiment 13 and/or 14.

16. An isolated cell comprising the vector of embodiment 15.

17. An isolated cell expressing the antibody as in any of embodiments1-12.

18. A method of producing an antibody comprising culturing the isolatedcell of embodiment 17 under conditions sufficient for the production ofthe antibody and recovering the antibody from the culture.

19. A pharmaceutical composition comprising the antibody as in any ofembodiments 1-12 in a pharmaceutically-acceptable carrier.

20. The pharmaceutical composition of embodiment 19, wherein theantibody is of the IgG1, IgG2, IgG3, or IgG4 human isotype.

21. A method of treating a B cell malignancy in a human comprisingadministering to a human in need thereof a therapeutically-effectiveamount of the antibody as in any of embodiments 1-12.

22. A method of treating an autoimmune disease or disorder in a human,comprising administering to a human in need thereof atherapeutically-effective amount of the antibody as in any ofembodiments 1-12.

23. A method of treating or preventing humoral rejection in a humantransplant patient, comprising administering to a human in need thereofa therapeutically-effective amount of the antibody as in any ofembodiments 1-12.

24. The antibody as in any of embodiments 1-12, wherein said antibodydepletes B cells with the same efficiency as that of the murinemonoclonal HB12B antibody.

25. The antibody as in any of embodiments 1-12, wherein said antibodyinduces B cell apoptosis.

26. The antibody as in any of embodiments 1-12, wherein said antibodyhas complex N-glycoside-linked sugar chains bound to the Fc region inwhich fucose is not bound to N-acetylglucosamine in the reducing end inthe sugar chain.

27. The antibody as in any of embodiments 1-12, wherein said antibody isan Fc variant antibody wherein said Fc variant comprises mutations thatresult in enhanced ADCC activity.

28. A method of depleting B cells in a human patient comprisingadministering to a human in need thereof a therapeutically-effectiveamount of the antibody as in any of embodiments 24-27.

29. The antibody as in any of embodiments 1-12, wherein said antibody isan Fc variant antibody wherein said Fc variant has an affinity for theFc receptor FcγRIIIA that is at least about 5 fold lower than that of acomparable molecule, wherein said Fc variant has an affinity for the Fcreceptor FcγRIIB that is within about 2 fold of that of a comparablemolecule.

30. A method of depleting B cells in a human patient comprisingadministering to a human in need thereof a therapeutically-effectiveamount of the antibody of embodiment 29.

7. EXAMPLES 7.1. Construction, Expression and Binding Characteristics ofHumanized Anti-CD19 Antibodies

The following sections describe the design and construction of achimeric variant of the parental HB12B antibody (chHB12B) in which themouse heavy chain and light chain constant regions have been replacedwith human IgH71 and human IgLK regions, respectively. These sectionsalso describe strategies for generation of humanized variants of HB12Bheavy and light chain variable regions.

The CD19-binding activity of antibodies produced from variouscombinations of (chimeric or humanized) heavy and light chain variableregions is also described. For example, humanized forms of HB12B whichexhibit a CD19 binding profile comparable to that of chHB12B aredescribed.

The sections below also describe several mutations in the humanframework regions that, when introduced into certain humanized anti-CD19antibodies, result in human CD19 binding comparable to that of thereference antibody, chHB12B comprising HB12B VH and HB12B VK. In the VKthese residues comprise, for example, the Vernier residues F36 and H49and Interchain residue F87.

7.1.1. Gene Assembly and Expression Cloning

Constructs were generated by a PCR-based gene assembly method firstdescribed by Stemmer (Stemmer, W. P. et al. 1995 Gene, 164:49-53). Thismethod consists of four steps: oligonucleotide synthesis; gene assembly;gene amplification and cloning. Eight gene specific primers weresynthesized for each VH and VK segments. Representative primer sets forthe assembly of the HB12B-(3-72/JH4) VH region and the HB12B-(A10-Jk4)VK are shown in Table 3; primer sets for variant VH and VK regionscomprising specific amino acid substitutions were generated by modifyingthe nucleic acid sequence of the primer encoding the given amino acidresidue. Primers were designed to overlap by 15-20 nucleotides and wereligated into a complete variable region during thermal cycling. In caseof VH, an additional vector specific primer (Universal VH FW in Table3.) was included in the PCR mediated gene assembly process. The external5′ and 3′ primers for VH region incorporated a unique recognition sitefor the Xbal and Apal restriction endonuclease, respectively, to helpwith the subsequent cloning steps. The external 5′ and 3′ primers for VKincorporated a unique recognition site for the XmaI and BsiWIrestriction endonuclease, respectively, to help with the subsequentcloning steps. PCR products of the correct size were restrictiondigested and ligated in frame into an expression vector wherein VHregions were digested with Xbal and Apal, and VK regions were digestedwith XmaI and BsiWI according to the manufacturer's instructions. Thevector used for heavy chain assembly comprises eukaryotic transcriptioncontrol elements operably linked to a polynucleotide encoding theMGDNDIHFAFLSTGVHS VH leader (SEQ ID NO:83) and a human IgHy1 constantregion wherein said transcription control elements comprise a CMVimmediate early promoter and a SV40 poly A addition signal. The use ofappropriately designed primers for VH assembly ensured that thepolynucleotide sequences encoding the VH leader, VH region and IgHy1constant region were joined in frame within the final heavy chainexpression vector. The vector for light chain assembly compriseseukaryotic transcription control elements operably linked to apolynucleotide encoding the human VKI-L12 leader (amino acid sequenceMDMRVPAQLLGLLLLWLPGAKC (SEQ ID NO:84); Bentley, D. L. & Rabbitts, T. H.,Nature 288, 730-733 (1980)) and a human IgLic constant region whereinsaid transcription control elements comprise a CMV immediate earlypromoter and a SV40 poly A addition signal. The use of appropriatelydesigned primers for VK assembly ensured that the polynucleotidesequences encoding the VKI-L12 leader, VK region and IgLic constantregion were joined in frame within the final light chain expressionvector. The ligation product was used to transform DH10B competent E.coli cells according to the manufacturer's protocols. Coloniescontaining the plasmid and a correct sized insert can be identifiedusing various methods known in the art (e.g. restriction digest ofvector DNA preparation, PCR amplification of vector sequences). Plasmidclones with correct sized insert were sequenced using dideoxy sequencingreaction (e.g., BigDye® Terminator v3.0 Cycle Sequencing Ready ReactionKit, ABI). Plasmid DNA was prepared from selected clones using theQIAGEN Mini and Maxi Plasmid Kit according to the manufacturer'sprotocols.

Pairs of DNA plasmid expression vector preparations encoding a humanizedor chimcric immunoglobulin heavy chain and a humanized or chimcricimmunoglobulin lightchain were used to co-transfect HEK293 cells. Theseco-transfected HEK293 cells were cultured for three days to yieldantibody-containing conditioned medium suitable for determining totalIgG concentrations and CD19 binding activity.

Total Ig concentrations in the HEK293 cell supernatant were quantifiedusing a capture ELISA assay. IgG molecules were captured on a 96-wellplate via an immobilized goat anti-human IgG H+L specific antibody, anddetected with an HRP conjugated anti-human kappa light chain antibody.The assay was calibrated using a reference IgG1 mAb of irrelevantspecificity.

TABLE 3Representative primer sets for HB12B-(3-72/JH4) VH region and theHB12B-(A1 0-Jk4) VK region assembly. Gene specific nucleotides areprinted in upper case, vector specific nucleotides are printed inlower case. Recognition sites for restriction endonucleases usedfor VH and VK fragment cloning are underlined. Universaltatatatatctagacatatatatgggtgacaatgacatccactttgcctttctctcc VH FW(SEQ ID NO: 85) HB12B-(3-GTTCATCCAAGAGCTACTGAAGGTGAATCCAGAGGCTGCACAGGAGAGTCTCAGGGACCCTCCAGGC72/JH4) RE2 (SEQ ID NO: 87) HB12B-(3-AGCTCTTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGGATTTATCCTGGAG72/JH4) FW3 (SEQ ID NO: 88) HB12B-(3-GCCCTTGAACTTCCCATTGTAGTTAGTATCTCCATCTCCAGGATAAATCCGGCCAACCCACTCCA72/JH4) RE4 (SEQ ID NO: 89) HB12B-(3-GGAAGTTCAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGTATCTGCAAATGAACAG72/JH4) FW5 (SEQ ID NO: 90) HB12B-(3-AATCCTGATCTAGCACAGTAATACACGGCCGTGTCCTCGGTTTTCAGGCTGTTCATTTGCAGATACAG72/JH4) RE6 (SEQ ID NO: 91) HB12B-(3-GTGTATTACTGTGCTAGATCAGGATTTATTACTACGGTTTTAGACTTTGACTACTGGG 72/JH4) FW7(SEQ ID NO: 92) HB12B-(3-tatatatagggcccttggtggaggcTGAGGAGACGGTGACCAGGGTTCCTTGGCCCCAGTAGTCAAAGTCTAAA72/JH4) RE8 (SEQ ID NO: 93) HB12B-(A10-tatatataccccggggccaaatgtGAAATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTG Jk4) FW1(SEQ ID NO: 94) HB12B-(A10-CAACACTTTCGCTGGCTCTGCAGGTGATGGTGACTTTCTCCTTTGGAGTCACAGACTGAAAGTCTGGJk4) RE2 (SEQ ID NO: 95) HB12B-(A10-GCCAGCGAAAGTGTTGATACTTTTGGCATTAGTTTTATGAACTGGTACCAGCAGAAACCAGATCAGTCJk4) FW3 (SEQ ID NO: 96) HB12B-(A10-CGAGGGGACCCCGGATCCTTGATTGGATGCAGCCTTGATGAGGAGCTTTGGAGACTGATCTGGTTTCJk4) RE4 (SEQ ID NO: 97) HB12B-(A10-GATCCGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCCTCACCATCAATAGCJk4) FW5 (SEQ ID NO: 98) HB12B-(A10-GAACCTCCTTACTTTGCTGACAGTAATACGTTGCAGCATCTTCAGCTTCCAGGCTATTGATGGTGAGGJk4) RE6 (SEQ ID NO: 99) HB12B-(A10-GCAAAGTAAGGAGGTTCCATTCACGTTCGGCGGAGGGACCAAGGTGGAGATCAAA Jk4) FW7(SEQ ID NO: 100) HB12B-(A10- tatatatacgtacgTTTGATCTCCACCTTGGTCCCTCCGCCGAJk4) RE8 (SEQ ID NO: 101)

7.1.2. The 300B4-CD19 Binding Assay

CD19 binding activity was assessed using a cell-based recombinant humanCD19 ELISA assay wherein said assay was performed using equivalentconcentrations of each humanized or chimeric antibody, therebyfacilitating direct comparisons between alternative humanized versionsof the HB12B antibody and chHB12B.

The ability of chHB12B and its humanized variants to bind hCD19 wasassessed in a cell based CD19 binding assay utilizing 300B4 cellsexpressing recombinant cell-surface human CD19 as a capture agent. 300B4cells were cultured according to standard protocols in RPMI 1640 mediumcontaining L-glutamine and supplemented with 10% Fetal Calf Serum,β-mercaptoethanol in the presence of 1 mg/ml G418. A standard ELISAprotocol can be used for the cell based CD19 binding assay. For example,individual wells of a 96 well U bottom plate are seeded with 1×10e5300B4 cells and incubated overnight. Cells are washed once with ELISAbuffer prior to incubation on ice with human, humanized, or chimericHB12B antibodies. Binding reactions are performed in triplicates foreach antibody concentration tested. Negative control wells using anisotype matched antibody of irrelevant specificity should be included inthe assay. Following incubation with the antibody 300B4 cells are washedthree times with 200 micro liter of ELISA buffer. The amount ofchimeric, humanized, or human anti-CD19 antibodies bound to 300B4 cellscan be detected using a goat anti-human kappa antibody conjugated withhorseradish peroxidase according to standard protocols.

7.1.3. Construction, Expression and Binding Characteristics of chHB12B

An expression vector encoding the chHB12B heavy chain comprising themurine HB12B VH and the human IgHγ1 constant regions was constructedaccording to the methods described in Section 6.1. An expression vectorencoding the chHB12B light chain comprising the murine HB12B VK and thehuman IgL_(K) constant regions was constructed according to the methodsdescribed in Section 6.1.

HEK293 cells were co-transfected simultaneously with the expressionvectors encoding chHB12B heavy and light chains. These transfected cellswere cultured for three days to allow for antibody production. Theculture medium containing soluble, secreted chHB12b antibody washarvested, and the concentration of chHB12B antibody was determinedaccording to the method described in Section 6.1.

chHB12B binding to human CD19 was assessed using the 300B4 cell basedELISA assay described in Section 6.2. An isotype matched human antibodyof irrelevant specificity was included in the assay as a negativecontrol. Results obtained using the cell based ELISA assay showed thatfor concentrations of antibody above 100 ng/ml, there was a significantbinding of chimeric antibody to recombinant human CD19 expressed on thesurface of 300B4 cells, indicating that the chHB12B has retained hCD19binding activity (FIG. 2).

7.1.4. Construction of Humanized HB12B VH Encoding Expression Vectors

The following sections describe the design of humanized variants of theHB12B VH region comprising the murine HB12B CDR regions and a suitablehuman, or substantially human framework regions. These sections alsodescribe strategies for generation of variants of humanized HB12B Igheavy chains.

7.1.4.1. Identification of Human Heavy Chain Acceptor Framework Regions

An amino acid sequence database containing the framework residues of allhuman germline immunoglobulin heavy chain V, D, and J regions werecompiled. The necessary information can be obtained from a variety ofsources, e.g. V Base: the database of human antibody genes(http://vbase.mrc-cpe.cam.ac.uk/). The database was queried for humangermline V and J segments that display sequence similarity with thecorresponding framework regions of the murine HB12b VH at key residues,e.g. canonical, interchain and Vernier residues.

The human germline V3-72 (Tomlinson, 1. M. et al., J. Mol. Biol.,227:776-798 (1992)) and JH4 (Ravetch, J. V. et al., Cell 27: 583-591(1981)) segments were selected to serve as the acceptor framework forthe humanization of the HB12B murine anti-CD19 antibody.

7.1.4.2. Generation of Humanized HB12B Heavy Chains

HB12B-(3-72/JH4) VH (SEQ ID NO:34) was designed by combining the CDRs ofHB12B VH with the framework residues of human germline V3-72/JH4regions. An expression vector comprising HB12B-(3-72/JH4) VH wasgenerated according to the methods described in Section 6.1.

HB12B-9m VH (SEQ ID NO:44) is a variant of HB12B-(3-72/JH4) VHcomprising the following nine amino acid substitutions: L20I, F27Y,T28A, R38I, V49I, F67A, R71A, L80M, I91Y (residues numbered according toKabat). Gene specific primers for HB12B-9m were designed as described inSection 6.1. An expression vector comprising HB12B-9m VH was generatedaccording to the methods described in Section 6.1.

7.1.5. Construction of Humanized HB12B Ig Light Chain EncodingExpression Vectors

The following sections describe the design of humanized variants of theHB12B VK region comprising murinc HB12B CDR regions and suitable human,or substantially human framework regions. These sections also describestrategies for generation of variants of humanized HB12B Ig light chain.

7.1.5.1. Identification of Human Light Chain Acceptor Framework Regions

An amino acid sequence database containing the framework residues of allhuman germline immunoglobulin light chain V and J regions were compiled.The necessary information can be obtained from a variety of sources,e.g. V Base: the database of human antibody genes(http://vbase.mrc-cpe.cam.ac.uk/). The database was queried for humangermline V and J segments that display sequence similarity with thecorresponding framework regions of the murine HB12B VK at key residues,e.g. canonical, interchain and Vernier residues.

The human germline Vk A10 (Straubinger, B. et al., Biol. Chem.Hoppe-Seyler 369:601-607 (1988)) and 0.11(4 (Hieter, P. A. et al., J.Biol. Chem. 257:1516-1522 (1982)) segments were selected to serve as theacceptor framework for the humanization of the HB12B murine anti-CD19antibody.

7.1.5.2. Generation of Humanized HB12B Light Chains

HB12B-(A10-Jk4) VK (SEQ ID NO:52) was designed by combining the HB12B VKCDRs with the framework residues of human germline A-10/Jk4 regions. Anexpression vector comprising HB12B-(A10-Jk4) VK was generated accordingto the methods described in Section 6.1.

HB12B-364987 VK (SEQ ID NO:62) is a variant of HB12B-(A10-Jk4) VKcomprising the following three amino acid substitutions: Y40F, K53H,Y91F (residues numbered according to Kabat). Gene specific primers forHB12B-364987 were designed as described in Section 6.1. An expressionvector comprising HB12B-364987 VK was generated according to the methodsdescribed in Section 6.1.

7.1.6. Binding Characteristics of Humanized HB12B Antibodies

Pairs of DNA plasmid expression vector preparations encoding (humanizedor chimeric) heavy and light immunoglobulin chains were used totransfect HEK293 cells. These transfected HEK293 cells were cultured forthree days to yield antibody-containing conditioned medium suitable fordetermining total IgG concentrations and CD19 binding activity.

Human CD19 binding activity of chimeric and humanized HB12B antibodieswas assessed using a 300B4 cell based ELISA assay described in Section6.2. An isotype matched human antibody of irrelevant specificity wasincluded in the assay as a negative control. As shown in FIG. 2, thebinding of a chimeric antibody comprising chHB12B heavy and chHB12Blight chains and the novel humanized antibody #1 comprisingHB12B-(3-72/JH4) VH and HB12B-364987 VK regions were found to becomparable. For concentrations of antibody above 100 ng/ml, there was asignificant specific binding of both antibodies to CD19, indicating thathumanized anti-CD19 antibody #1 comprising HB12B-(3-72/JH4) VH andHB12B-364987 VK regions has retained CD19 binding activity.Surprisingly, humanized antibodies comprising the HB12B-9m VH regiondisplayed a significant loss in CD19 compared to chHB12B control.

Pairs of chimeric or humanized HB12B heavy and light chains were testedand their human CD19 binding activity is summarized in Table 4.

TABLE 4 CD19 binding of chimeric and humanized HB12B antibodies. CD19binding activity of various chimeric and humanized HB12B antibodies wasassessed using a cell based ELISA assay. VH-VK combinations displayingsignificant binding activity are marked with “++”. VH-VK combinationswith no significant binding to human CD19 are marked with “−”. HB12B-chHB12B VH (3-72/JH4) VH HB12B-9m VH chHB12B VK ++ ++ − HB12B-(A10-Jk4)VK − − − HB12B-364987 VK ++ ++ −

7.1.7. Construction, Expression and Binding Characteristics of HumanizedHB12B Light Chains

Antibody humanization protocols generally try to limit the number ofnon-human framework residues in order to minimize the HAMA response.Accordingly, additional variants of humanized HB12B VK were generatedand their hCD19 binding activity was assessed.

HB12B-3649 VK (SEQ ID NO:68) is a variant of HB12B-(A10-Jk4) VKcomprising the following two amino acid substitutions: Y40F, K53H(numbering according to Kabat). An expression vector comprisingHB12B-3649 VK was generated via site directed mutagenesis using theQuickChange kit (Stratagene, La Jolla, Calif.) on a DNA preparation ofan expression vector comprising HB12B-364987 VK according to themanufacturer's instruction.

HB12B-3687 VK (SEQ ID NO:74) is a variant of HB12B-(A10-Jk4) VKcomprising the following two amino acid substitutions: Y40F, and Y91F(numbering according to Kabat). An expression vector comprisingHB12B-3687 VK was generated via site directed mutagenesis using theQuickChange kit (Stratagene, La Jolla, Calif.) on a DNA preparation ofan expression vector comprising HB12B-364987 VK according to themanufacturer's instruction.

HB12B-4987 VK (SEQ ID NO:76) is a variant of HB12B-(A10-Jk4) VKcomprising the following two amino acid substitutions: K53H, and Y91F(numbering according to Kabat). An expression vector comprisingHB12B-4987 VK was generated via site directed mutagenesis using theQuickChange kit (Stratagene, La Jolla, Calif.) on a DNA preparation ofan expression vector comprising HB12B-364987 VK according to themanufacturer's instruction.

HB12B-36 VK (SEQ ID NO:70) is a variant of HB12B-(A10-Jk4) VK comprisingthe following acid substitution: Y40F (numbering according to Kabat). Anexpression vector comprising HB12B-36 VK was generated via site directedmutagenesis using the QuickChange kit (Stratagene, La Jolla, Calif.) ona DNA preparation of an expression vector comprising HB12B-(A10-Jk4) VKaccording to the manufacturer's instruction.

HB12B-49 VK (SEQ ID NO:80) is a variant of HB12B-(A10-Jk4) VK comprisingthe following amino acid substitution: K53H (numbering according toKabat). An expression vector comprising HB12B-49 VK was generated viasite directed mutagenesis using the QuickChange kit (Stratagene, LaJolla, Calif.) on a DNA preparation of an expression vector comprisingHB12B-(A10-Jk4) VK according to the manufacturer's instruction.

HB12B-87 VK (SEQ ID NO:78) is a variant of HB12B-(A10-Jk4) VK comprisingthe following amino acid substitution: Y91F (numbering according toKabat). An expression vector comprising HB12B-87 VK was generated viasite directed mutagenesis using the QuickChange kit (Stratagene, LaJolla, Calif.) on a DNA preparation of an expression vector comprisingHB12B-(A10-Jk4) VK according to the manufacturer's instruction.

Pairs of DNA plasmid expression vector preparations encoding a heavychain comprising HB12B-(3-72/JH4) VH and each of the VK variantsdescribed in Section 6.1.7. were used to co-transfect HEK293 cells.These co-transfected HEK293 cells were cultured for three days to yieldhumanized antibody-containing conditioned medium suitable fordetermining total IgG concentrations and hCD19 binding activity.

Human CD19 binding activity of humanized HB12B antibodies was assessedusing a 300B4 cell based ELISA assay described in Section 6.1.2. chHB12Bwas included in the assay as a positive control. As shown in FIG. 3, thebinding of chHB12B antibody comprising chHB12B VH and chHB12B VK and thenovel humanized HB12B antibody #2 comprising HB12B-(3-72/JH4) VH andHB12B-3649 VK were found to be comparable. For concentrations ofantibody above 100 ng/ml, there was a significant specific binding ofboth antibodies to hCD19, indicating that the humanized antibodycomprising HB12B-(3-72/JH4) VH and HB12B-3649 VK retained hCD19 bindingactivity. Humanized HB12B antibody #1 comprising HB12B-364987 VK alsodisplayed binding of human CD19. Humanized HB12B antibody #3 comprisingHB12B-36 VK exhibited significantly reduced binding to hCD19 compared tothe binding of the chHB12B control antibody.

Taken together, these data indicate that a number of humanized versionsof the HB12B VH and VK chains were created that retains the bindingproperties of the parental mouse antibody derived from the HB12Bhybridoma.

7.2. In Vitro ADCC Activity of Humanized Anti-CD19 Antibodies

The following sections describe the characterization of the in vitroADCC activity of humanized anti-CD19 antibodies.

7.2.1. Humanized Anti-CD19 Antibody Preparations.

Purified humanized anti-CD19 antibody #2 comprising HB12B-(3-72/JH4) VH,HB12B-3649 VK, and IgG1 heavy chain constant region (hereinafterreferred to as “3649 antibody” or “3649”) is prepared using standardtechniques. Briefly, a DNA plasmid expression vector preparationencoding the heavy and light chains of 3649 is used to transfect HEK293Fcells. Transfected cells are fed at day 3 and 6 and theantibody-containing conditioned medium is harvested at day 9. Antibodyis purified from the conditioned medium using a pre-cast protein Acolumn (GE Healthcare). Antibody is eluted from the column with low pHbuffer, neutralized, and dialyzed against PBS. The concentration of thepurified antibody is calculated from the solution's optical density at280 nm.

An antibody expression vector encoding a 3649 Fc variant comprisingS239D, A330L, and I332E amino acid substitutions (hereinafter referredto as “3649-3M”) is generated using methods described in US 2004/0132101and US 2005/0054832, both to Lazar et al. Briefly, the antibodyexpression vector encoding 3649 is modified using a site directedmutagenesis kit (e.g., QuickChange (Promega)) by introducing thenecessary nucleotide residue substitutions into the polynucleotidesequence encoding the heavy chain constant region to generate the3649-3M antibody expression vector. Purified 3649-3M antibody isgenerated by transfecting HEK239F cells with the 3649-3M antibodyexpression vector. Transfected cells are fed at day 3 and 6 and theantibody-containing conditioned medium is harvested at day 9. Antibodyis purified from the conditioned medium using a pre-cast protein Acolumn (GE Healthcare). Antibody is eluted from the column with low pHbuffer, neutralized, and dialyzed against PBS. The concentration of thepurified antibody is calculated from the solution's optical density at280 nm.

An antibody expression vector encoding a 3649 Fc variant comprising theL234F, L235E, and P331S amino acid substitutions (hereinafter referredto as “3649-TM”) is generated using methods described in US 2004/0132101and US 2005/0054832, both to Lazar et al., each of which is incorporatedherein by reference in their entirety. Briefly, the antibody expressionvector encoding 3649 is modified using a site directed mutagenesis kit(e.g., QuickChange (Promega)) by introducing the necessary nucleotideresidue substitutions into the polynucleotide sequence encoding theheavy chain constant region to generate the 3649-TM antibody expressionvector. Purified 3649-TM antibody is generated by transfecting HEK239Fcells with the 3649-TM antibody expression vector. Transfected cells arefed at day 3 and 6 and the antibody-containing conditioned medium isharvested at day 9. Antibody is purified from the conditioned mediumusing a pre-cast protein A column (GE Healthcare). Antibody is elutedfrom the column with low pH buffer, neutralized, and dialyzed againstPBS. The concentration of the purified antibody is calculated from thesolution's optical density at 280 nm.

A 3649 antibody composition (hereinafter referred to as 3649-aFuc)comprising a plurality of antibodies having complex N-glycoside-linkedsugar chains linked to Asn297 of the Fc region in which fucose is notbound to N-acetylglucosamine in the reducing end was prepared accordingto the methods set forth in U.S. Pat. No. 6,946,292 to Kanda et al.,which is incorporated herein by reference in its entirety. Briefly,fucosyltransferase knock-out CHO cells are transfected with a DNAplasmid expression vector preparation encoding the heavy and lightchains of 3649. Transfected cells are fed at day 3 and 6 and theantibody-containing conditioned medium is harvested at day 9. Antibodyis purified from the conditioned medium using a pre-cast protein Acolumn (GE Healthcare). Antibody is eluted from the column with low pHbuffer, neutralized, and dialyzed against PBS. The concentration of thepurified antibody is calculated from the solution's optical density at280 nm.

Antibody preparations were substantially pure from contaminatingproteins as demonstrated in FIG. 4. Antigen binding affinity of3649-aFuc is comparable to that of 3649 as shown in FIG. 5.

7.2.2. In Vitro ADCC Assay

The CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) is acolorimetric alternative to ⁵¹Cr release cytotoxicity assays. TheCytoTox 96® Assay quantitatively measures lactate dehydrogenase (LDH), astable cytosolic enzyme that is released upon cell lysis. Released LDHin culture supernatants is measured with a 30-minute coupled enzymaticassay, which results in the conversion of a tetrazolium salt (INT) intoa red formazan product. The amount of color formed is proportional tothe number of lysed cells.

The assays are performed according to the manufacturer's directions.Briefly, target cells are washed with PBS, resuspended in RPMI-5 PhenolFree media at a cell density of 0.4×10⁶/ml. NK effector cells are washedonce in PBS and resuspended in RPMI-5 Phenol Free media at a celldensity 1×10⁶/ml. Assays are performed in U bottom 96 well plates. Eachassay plate includes a combination of experimental and control wells.Experimental wells are set up by combining 50 μl of the appropriateantibody dilution, 50 μl of target cell suspension and 50 μl of effectorcell suspension. The cell densities described above result in a 1:2.5target to effector cell ratio; effector cell stock may be furtherdiluted or concentrated if a different target to effector ratio isdesired. Several different types of control wells are used to accountfor (i) the spontaneous LDH release form target cells (TargetSpontaneous), (ii) the spontaneous LDH release from effector cells(Effector Spontaneous), (iii) the maximum LDH release from the targetcells (Target Maximum), and (iv) the presence of contaminants in theculture medium (Background). All wells in use on a 96 well plate containthe same final volume. Reactions are set up in triplicates. Followingset up, plates are spun at 120×g for 3 minutes to pellet the cells.Incubate plate at 37° C./5% CO₂ for 4 hours. Forty five minutes prior tothe end of incubation 15 μl of manufacturer provided Lysis Buffer isadded to the Target Cell Maximum Release Control well. After incubationthe plate is centrifuged at 120×g for 4 minutes. 50 μl of thesupernatant from each well is transferred to a new flat bottom 96 wellplate. 50 p.1 of reconstituted substrate mix (assembled frommanufacturer provided components) is added and the plate is incubated atroom temperature 10-20 minutes protected from light. 50 μl ofmanufacturer provided stop buffer is added and absorbance at 490 or 492nm is measured in a plate reader. % cytotoxicity equals(Experimental−Effector spontaneous−Target Spontaneous)/(TargetMaximum−Target Spontaneous). Prior to calculating the % cytotoxicity allother values are reduced by the Background.

3649 efficiently recruits effector cells to human CD20 expressing targetcells in an ADCC assay. The ADCC activity of the afucosylated form(3649-aFuc) is even more robust. Fc variants with increased (3649-3M) ordecreased (3649-TM) affinity for Fcγ receptors display increased ordecreased, respectively, ADCC activity as expected. ADCC activity wasobserved with both immortalized and freshly isolated human target cells.A representative sample of the experimental data supporting theseassertions is presented in FIGS. 5 to 9.

7.2.3. In Vitro Anti-CD19 Antibody Mediated ADCC is Influenced by FcRegion Affinity to FcγRIIIA Receptor.

Relative binding affinity of various humanized anti-CD19 antibodypreparations to human FcγRIIIA receptor (CD16) may be ascertained usingan ELISA assay. Microtiter plates are coated with 50 pi antibodypreparation (50 μg/ml) at 4° C. overnight. Any remaining binding sitesare blocked with 4% skimmed milk in PBS buffer (blocking buffer) for 1 hat 37° C. After washing the wells, 50 μl of serially diluted monomericFcγRIIIA-flag protein is added to each well and incubated for 60 min at37° C. 50 μl of 2.5 μg/ml anti-flag-ME-biotin (Sigma) is added to eachwell and incubated for 30 min at 37° C. Wells are washed betweenincubation with each of the following reagents. 50 μl of 0.1 μg/mlavidin-conjugated HRP (PIERCE) is added to each well and incubated for30 min at 37° C. Detection is carried out by adding 30 μl μl oftetramethylbenzidine (TMB) substrate (Pierce) followed by neutralizationwith 30 μl of 0.2 M H2504. The absorbance was read at 450 nm.

As shown in FIG. 15, the binding affinity of the enhanced ADCC Fcvariant 3649 antibody (3649-3M) and afucosylated 3649 antibody(3649-aFuc) for FcgRIIIA is higher than that of the fucosylated wildtype 3649 antibody. The experiment was performed using an FcγRIIIA-flagprotein comprising the extracellular domain of the V158 high affinityisoform of human FcγRIIIA

The Fc receptor-Fe region interaction, and thus the effector function ofan antibody, are also influenced by allelic variations in the Fcreceptor. The effect of high affinity and low affinity FcgRIIIAreceptors on ADCC may be studied by performing ADCC reactions withfreshly isolated NK effector cells comprising different allelic variantreceptors. FIG. 16 summarizes the results of such an experiment. ADCCreactions are performed as described above using Daudi target cells.Both fucosylated (3649) and afucosylated (3649-aFuc) anti-CD19 antibody#2 is tested. Control reactions are done using an anti-CD20 antibody. NKeffector cells are isolated from healthy donors following standardprotocols. NK cell genotype may be determined utilizing allele specificPCR reactions (see, Leppers-van de Straat et al., J Immunol Methods.242(1-2):127-32 (2000)). FIGS. 16 A and B show that all three antibodiestested display ADCC activity under the reaction conditions used. TheADCC activity is detectable using either an NK cell line (A) or freshlyisolated NK cells (B) as effectors. NK cells that comprise at least onecopy of the high affinity isoform of FcgRIIIA receptor (V158/V158 andV158/F158 genotypes) are more efficient effector cells than NK cellshomozygous for the low affinity receptor alleles (F158/F158 genotype)(FIG. 16C-E). Lack of fucosylation increases the ADCC activity of anantibody regardless of the FcgRIIIA genotype of the effector cells. Theobserved ADCC activity of the fucosylated antibody (3649) mediated byV158/V158 or V158/F158 NK cells (C, D) is comparable to the ADCCactivity of the afucosylated antibody (3649-aFuc) mediated by F158/F158NK cells (E).

7.3. Antibodies and Immunofluorescence Analysis

Anti-CD19 antibodies described above, which bind to the human CD19antigen, can be used in the approaches disclosed below. Otherantibodies, which could be employed in the experiments described belowinclude monoclonal mouse anti-CD22 antibodies that bind to mouse CD22,e.g. HIB22 (Abeam; Dorken B et al., J Immunol 136:4470-9 (1986));monoclonal mouse CD20-specific antibodies (Uchida et al., Intl. Immunol.16:119-129 (2004)); B220 antibody RA3-6B2 (DNAX Corp., Palo Alto,Calif.); and CD5, CD43 and CD25 antibodies (BD PHARMINGEN™, FranklinLakes, N.J.). Isotype-specific and anti-mouse Ig or IgM antibodies canbe obtained from Southern Biotechnology Associates, Inc. (Birmingham,Ala.).

Either mouse pre-B cell lines, transfected with hCD19 cDNA, which can bedeveloped using methods and materials known in the art (see e.g. Alt etal., Cell, 27:381-388 (1981) and Tedder and Isaacs, J. Immunol.,143:712-717 (1989)), or single-cell leukocyte suspension, are stained onice using predetermined, optimal concentrations of eachfluorescently-labeled antibody for 20-30 minutes according toestablished methods (Zhou et al., Mol. Cell. Biol., 14:3884-3894(1994)). Cells with the forward and side light scatter properties oflymphocytes can then be analyzed on FACSCAN® or FACSCALIBUR® flowcytometers (Becton Dickinson, San Jose, Calif.). Background stainingwould be determined using unreactive control antibodies (CALTAG™Laboratories, Burlingame, Calif.) with gates positioned to excludenonviable cells. For each sample examined, ten thousand cells with theforward and side light scatter properties of mononuclear cells areanalyzed whenever possible, with fluorescence intensities shown on afour-decade log scale.

Mice.

Transgenic mice expressing hCD19 and their wild-type (WT) littermatescan be produced as described in the art (Zhou et al., Mol. Cell. Biol.,14:3884-3894 (1994)). For example, hCD19tg mice can be generated fromoriginal hCD19 founders (e.g. C57BL/6×B6/SJL), and then crossed onto aC57BL/6 background for at least 7 generations. After multiplegenerations of backcrossing, mice would be obtained in which their Bcells would express cell surface density of human CD19 at about the samedensity found on human B cells.

Mice bred with FcR (Fe receptor) common γ chain (FcRγ)-deficient mice(FcRγ−/−, B6.129P2-Fcerg1^(tm1)) are available from Taconic Farms(Germantown, N.Y.) and could be used to generate hCD19 FcRγ−/− and WTlittermates. Mice hemizygous for a c-Myctransgene (Eμ-cMycTG,C57B1/6J-TgN(IghMyc); The Jackson Laboratory, Bar Harbor, Me.) aredescribed in the art (Harris et al., J. Exp. Med., 167:353 (1988) andAdams et al., Nature, 318:533 (1985)). c-MycTG mice (B6/129 background)could be crossed with hCD19tg mice to generate hemizygous hCD19tgcMycTG′i-offspring that could be identified by PCR screening. Rag1(B6.129S7—Rag1^(tmlMom)/J) mice are available from The JacksonLaboratory. Macrophage-deficient mice can be generated by tail veininjections of clodronate-encapsulated liposomes (0.1 mL/10 gram bodyweight; Sigma Chemical Co., St. Louis, Mo.) into C57BL/6 mice on day −2,1 and 4 in accordance with standard methods (Van Rooijen and Sanders, J.Immunol. Methods, 174:83-93 (1994)). All mice should be housed in aspecific pathogen-free barrier facility and first used at 6-9 weeks ofage.

ELISAs.

Serum Ig concentrations are determined by ELISA using affinity-purifiedmouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA (Southern BiotechnologyAssociates, Inc., Birmingham, Ala.) to generate standard curves asdescribed (Engel et al., Immunity, 3:39 (1995)). Serum IgM and IgGautoantibody levels against dsDNA, ssDNA and histone are determined byELISA using calf thymus double-stranded (ds) DNA (Sigma-Aldrich, St.Louis, Mo.), boiled calf thymus DNA (which contains single-stranded (ss)DNA) or histone (Sigma-Aldrich) coated microtiter plates as described(Sato et al., J. Immunol., 157:4371 (1996)).

Immunotherapy.

Sterile anti-CD19 and unreactive, isotype control antibodies (0.5-250μg) in 200 μL phosphate-buffered saline (PBS) are injected throughlateral tail veins. For example, experiments would use a fixed amount(e.g. 250 μg) of antibody. Blood leukocyte numbers are quantified byhemocytometer following red cell lysis, B220⁺ B cell frequencies aredetermined by immunofluorescence staining with flow cytometry analysis.Antibody doses in humans and mice would be compared using the OncologyTool Dose Calculator.

Immunizations.

Two-month old WT mice are immunized i.p. with 50 μg of2,4,6-trinitrophenyl (TNP)-conjugated lipopolysaccharide (LPS) (Sigma,St. Louis, Mo.) or 25 μg 2,4-dinitrophenol-conjugated (DNP)-FICOLL®(Biosearch Technologies, San Rafael, Calif.) in saline. Mice are alsoimmunized i.p. with 100 μg of DNP-conjugated keyhole limpet hemocyanin(DNP-KLH, CALBIOCHEM®-NOVABIOCHEM® Corp., La Jolla, Calif.) in completeFreund's adjuvant and are boosted 21 days later with DNP-KLH inincomplete Freund's adjuvant. Mice are bled before and afterimmunizations as indicated. DNP- or TNP-specific antibody titers inindividual serum samples are measured in duplicate using ELISA platescoated with DNP-BSA (CALBIOCHEM®-NOVABIOCHEM® Corp., La Jolla, Calif.)or TNP-BSA (Biosearch Technologies, San Rafael, Calif.) according tostandard methods (Engel et al., Immunity, 3:39-50 (1995)). Sera fromTNP-LPS immunized mice are diluted 1:400, with sera from DNP-FICOLL® andDNP-BSA immunized mice diluted 1:1000 for ELISA analysis.

Statistical Analysis.

All data would be shown as means±SEM with Student's t-test used todetermine the significance of differences between sample means

7.4. Human CD19 Expression in Transgenic Mice

Transgenic hCD19tg mice, which can be developed as described herein, orother transgenic animals expressing human CD19 can be used to assessdifferent therapeutic regimens comprising anti-CD19 antibodies, such asvariations in dosing concentration, amount, and timing. The efficacy inhuman patients of different therapeutic regimens can be predicted usingthe two indicators described below, i.e., B cell depletion in certainbodily fluids and/or tissues and the ability of a monoclonal human orhumanized anti-CD19 antibody to bind B cells. In particular embodiments,treatment regimens that are effective in human CD19 transgenic micecould be used with compositions and methods of the invention to treathuman B cell disorders and disease including, but not limited to, B cellmalignancies and autoimmune diseases or disorders.

In order to determine whether human CD19 is expressed on B cells fromtransgenic mice (hCD19tg) expressing the human CD19 transgene, B cellswould be extracted from the bone marrow, blood, spleen and peritoneallavage of these mice. Human CD19 and mouse CD19 expression would beassessed in these cells by contacting the cells with anti-CD19antibodies that specifically bind human CD19 or mouse CD19 (mCD19).Binding of the antibody to the B lineage cells would be detected usingtwo-color immunofluorescence staining with flow cytometry analysis. Therelative expression levels of mCD19 and hCD19, would be assessed bymeasuring mean fluorescence intensity (anti-hCD19 for hCD19 andanti-mCD19 for mCD19) respectively.

Expression level of a human CD19 and human CD20 transgenes wasdetermined essentially as described above. Circulating lymphocytes areisolated form C57B16 hCD19 tg+/−, C57B16 hCD19 tg+/+, Balb/c hCD20 tg+/−and Balb/c wild type mice using standard procedures. Animals were housedin a pathogen free facility. The age and number of animals used fromeach genotype is listed in FIG. 10. Isolated cells are stained withPerCP Cy5.5 conjugated anti-mouse CD19 (clone 1D3, BD Biosciences), PEconjugated anti-CD3 (e.g., clone 17A2, BD Biosciences), Alexa Fluor® 488conjugated anti-human CD19 (clone HIB19, BD Biosciences), and AlexaFluor® 647 conjugated anti-human CD20 antibodies (e.g., clone 2H7, AbDserotec). Immunostained cells are analyzed on a flow cytometer. B cellpopulation is defined as anti-mouse CD19+, anti-CD3− cells. Meanfluorescence intensity of anti-mouse CD19+, anti-CD3− cells detected inthe hCD19 and hCD20 channels is described in FIG. 10A. Human CD19expression is detected only on hCD19 transgenic cells as expected. hCD19expression is dose dependent; staining levels in tg+/+ is approximatelytwice that of seen on tg+/−B cells. hCD19 expression level was stable inall age groups examined.

Percentage of B cells among circulating lymphocytes was calculated forall samples. B cells were defined as anti-mouse CD19+, anti-CD3− cellsfor the purpose of the calculation. Results are displayed in FIG. 10B.Animals with a hCD19 transgene have reduced B cell numbers amongcirculating lymphocytes. Reduction in B cell numbers are more pronouncedin hCD19 tg+/+ animals. These results are in agreement with previouslypublished observations (Zhou et al., Mol. Cell. Biol., 14:3884-3894(1994)).

7.5. Anti-CD19 Antibody Mediated Depletion of B Cells In Vivo

Anti-CD19 antibodies of the invention, which bind to human CD19, can beassessed for their ability to deplete hCD19tg blood, spleen, and lymphnode B cells in vivo. For example, each antibody would be given to miceat either 250 or 50 μg/mouse, a single dose about 10 to 50-fold lowerthan the 375 mg/m² dose primarily given four times for anti-CD20 therapyin humans (Maloney et al., J. Clin. Oncol., 15:3266-74 (1997) andMcLaughlin et al., Clinical status and optimal use of rituximab for Bcell lymphomas, Oncology (Williston Park), 12:1763-9 (1998)). B celldepletion from blood, spleen and lymph nodes of hCD19tg mice would bedetermined by immunofluorescence staining with flow cytometry analysis.The results using anti-CD19 antibodies identified as capable ofdepleting B cells can be correlated to use in humans and antibodies withproperties of the identified antibodies can be used in the compositionsand methods of the invention for the treatment of human B cell disordersand disease including, but not limited to, B cell malignancies andautoimmune diseases or disorders.

3649 humanized anti-CD19 antibody was tested in a B cell depletion assayessentially as described above. C57B16 hCD19 tg+/− and C57B16 hCD19tg+1+ mice are given a single i.v. dose of 50 or 250 iLig 3649 antibody.Two control groups are used. Members of the first group receive 50 or250 jag of R347 antibody of irrelevant specificity; members of thesecond group receive 50 or 250 μg of the 3649-TM Fc variant withdiminished ADCC activity (see FIG. 6). Number of animals in each groupare described in FIGS. 11 and 12. Animals are housed in a pathogen freefacility. 7 days post treatment mononuclear cells are isolated fromcirculating blood and spleens. Isolated cells are stained with PerCPCy5.5 conjugated anti-mouse CD19 (clone 1D3, BD Biosciences) andApc-Cy5.5 conjugated anti-mouse B220 (cloneRA3-6B2, Invitrogen)antibodies. Immunostained samples are analyzed on a flow cytometer. Bcells are defined as anti-mouse CD19+, anti-mouse B220+ cells for thepurposes of the experiment. The percentage of B cells among circulatinglymphocytes is presented in FIG. 11. The percentage and absolute numberof B cells among spleen cells is described in FIG. 12. A single dose of50 mg 3649 anti-CD19 antibody is sufficient to achieve significantdepletion of circulating and splenic B cells. Level of depletion isinfluenced by antibody dose and hCD19 surface density. Depletion is mostcomplete in hCD19 tg+/+ animals receiving 250 μg antibody. Depletion ismore extensive among circulating lymphocytes than in spleen in allanimals tested.

A separate study was also performed to measure the ability of various3649 anti-CD19 antibody preparations to deplete circulating, splenic,peritoneal and bone marrow B cell subpopulations in a hCD19 tg+/−animal. The experiment was performed as follows: Three to four-month oldsex matched C57B16 hCD19 tg+/− mice were injected through the lateraltail veins with sterile endotoxin-free anti-CD19 antibody preparationsdiluted in PBS at 10, 50 or 250 ug per mouse doses. The followinganti-CD19 antibodies were tested: fucosylated anti-CD19 antibody #2(3649), afucosylated anti-CD19 antibody #2 (3649-aFuc), ADCC enhanced Fcvariant anti-CD19 antibody #2 (3649-3M), and reduced ADCC Fc variantanti-CD19 antibody #2 (3649-TM). A group of control animals wereinjected with an isotype matched control antibody of irrelevantspecificity (R347). Seven days post injection, mice were scarified andcells from blood, spleen, bona marrow and peritoneal cavity werecollected. Red cells were lysed following standard protocols and totalviable cell count was determined using a Via-Cell automated cellcounting machine. Isolated single cell suspensions were immunostainedand analyzed on a flow cytometer following standard protocols.Antibodies used for immunostaining are listed in Table 5. Depletionresults are summarized in Tables 6¬21. Depletion results obtained usingthe afucosylated anti-CD19 antibody #2 (3649-aFuc) is presented in FIG.28. NK cell activation phenotype of animals treated with theafucosylated or fucosylated anti-CD19 antibody #2 (3649-aFuc or 3649,respectively) is presented in FIG. 29. B cell subset definitions usedduring the analysis are as follows:

-   Blood: B cells: B220+, mouse CD19+-   Spleen: B cells: B220+, mouse CD19+    -   Transitional B cells: after gating on B cells, CD93+        -   Transitional 1 B cells (T1): IgM+CD23−        -   Transitional 1 B cells (T2): IgM+CD23+        -   Transitional 1 B cells (T3): IgMlowCD23+    -   Mature B cells: after gating on B cells, CD93−        -   Follicular B cells: IgM+CD23+        -   Marginal Zone B cells: IgMhighCD23−-   Bone Marrow: B cells: B220+, mouse CD19+    -   Pro-B cells: after gating on B cells, CD43+IgM−    -   Pre-B cells: after gating on B cells, CD43−IgM−    -   Immature and Mature B cells: after gating on B cells, CD43−IgM+        -   Immature B cells: CD43−IgM+CD93+        -   Mature B cells: CD43−IgM+CD93+low/−-   Peritoneal Cavity: B cells: IgM+

TABLE 5 Antibodies used for B cell identification in in vivo depletionexperiments. Dead cells were detected by 7-AAD staining Clone AntigenDye Number Source Spleen B220 FITC RA3-6B2 BD Bioscience CD19 PerCP- 1D3BD Cy5.5 Bioscience CD23 PE B3B4 Biolegend hCD19 PE-Alexa SJ25-C1Invitrogen 610 IgM PE-Cy7 II/41 eBioscience C1qRp Apc AA4.1 eBioscienceBone B220 FITC RA3-6B2 BD Marrow Bioscience CD19 PerCP- 1D3 BD Cy5.5Bioscience CD43 PE S7 BD Bioscience hCD19 PE-Alexa SJ25-C1 Invitrogen610 IgM PE-Cy7 II/41 eBioscience C1qRp Apc AA4.1 eBioscience PeritonealB220 FITC RA3-6B2 BD Cavity Bioscience CD19 PerCP- 1D3 BD Cy5.5Bioscience CD43 PE S7 BD Bioscience hCD19 PE-Alexa SJ25-C1 Invitrogen610 IgM PE-Cy7 II/41 eBioscience CD5 Apc 53-7.3 eBioscience Blood hCD19Alexa 488 SJC25 Invitrogen CD3 PE mCD19 PerCP Cy5.5 1D3 BD BioscienceB220 Alexa 647 RA3-6B2 BD Bioscience

TABLE 6 Summary of circulating B cell depletion results. 3649, 3649-3M,3649-TM or control R347 antibody was administered to hCD19tg+/− micefollowing the protocol described above. % B cell is defined as theB220+, mouse CD19+ fraction of blood lymphocytes; lymphocyte populationis detected based on characteristic forward and side scatter profile(see FIG. 17A for details). % depletion was calculated as 100 × (% Bcell (control antibody) − % B cell(experimental antibody))/% B cell(control antibody). Negative depletion numbers are used when the size ofa cell population in the treated animal was larger than thecorresponding size in the control animal. Dose Antibody (μg/mouse) % Bcell % Depletion R347 10 26.8% N/A R347 50 37.9% N/A R347 250 22.2% N/A3649 10 16.9% 36.94% 3649 50 1.5% 96.12% 3649 250 1.0% 95.64% 3649-3M 101.1% 95.82% 3649-3M 50 0.1% 99.84% 3649-TM 50 32.8% 13.47% 3649-TM 25026.4% (−18.7%)

TABLE 7 Summary of splenic B cell depletion results. 3649, 3649-3M,3649-TM, or control R347 antibody was administered to hCD19tg+/− micefollowing the protocol described above. % B cell is defined as theB220+, mouse CD19+ fraction of lymphocytes (see FIG. 17B for details). %depletion was calculated as 100 × (cell number (control antibody) − cellnumber (experimental antibody))/cell number (control antibody). Negativedepletion numbers are used when the size of a cell population in thetreated animal was larger than the corresponding size in the controlanimal. Dose Antibody (μg/mouse) % B cell Cell number/animal % DepletionR347 10 17.2% 5,991,451 N/A R347 50 22.5% 4,620,997 N/A R347 250 25.7%5,317,874 N/A 3649 10 13.4% 3,267,904 45.5% 3649 50 6.8% 773,147 83.3%3649 250 7.8% 947,293 82.2% 3649-3M 10 4.1% 532,244 91.1% 3649-3M 501.6% 102,285 97.8% 3649-TM 50 28.5% 6,199,144 (−34.1%) 3649-TM 250 21.8%4,182,489 21.4%

TABLE 8 Summary of splenic transitional B cell depletion results. 3649,3649-3M, 3649-TM, or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. % transitional Bcells are defined as the CD93+ fraction of B cells (see FIG. 17B fordetails). % depletion was calculated as 100 × (cell number (controlantibody) − cell number (experimental antibody))/cell number (controlantibody). Dose % transitional Cell number/ Antibody (μg/mouse) B cellsanimal % Depletion R347 10 17.4% 1,131,356 N/A R347 50 18.4% 937,279 N/AR347 250 17.0% 975,450 N/A 3649 10 11.1% 402,770 64.4% 3649 50 3.0%24,173 97.4% 3649 250 4.0% 39,299 96.0% 3649-3M 10 7.2% 43,106 96.2%3649-3M 50 5.2% 5,272 99.4% 3649-TM 50 12.4% 840,608 10.3% 3649-TM 25010.2% 455,248 53.3%

TABLE 9 Summary of splenic T1 B cell depletion results. 3649, 3649-3M,3649-TM, or control R347 antibody was administered to hCD19tg+/− micefollowing the protocol described above. Ti B cells are defined as theIgM+, CD23− fraction of transitional B cells (see FIG. 17B for details).% depletion was calculated as 100 × (cell number (control antibody) −cell number (experimental antibody))/cell number (control antibody).Dose % T1 B Cell number/ % Antibody (μg/mouse) cells animal DepletionR347 10 28.8% 335,185 N/A R347 50 36.0% 332,727 N/A R347 250 34.6%367,735 N/A 3649 10 27.2% 107,298 68.0% 3649 50 23.3% 5,845 98.2% 3649250 30.4% 12,678 96.6% 3649-3M 10 24.2% 10,438 96.9% 3649-3M 50 20.4%1,168 99.6% 3649-TM 50 29.4% 253,730 23.7% 3649-TM 250 27.3% 125,49165.9%

TABLE 10 Summary of splenic T2 B cell depletion results. 3649, 3649-3M,3649-TM, or control R347 antibody was administered to hCD19tg+/− micefollowing the protocol described above. T2 B cells are defined as theIgM+, CD23+ fraction of transitional B cells (see FIG. 17B for details).% depletion was calculated as 100 × (cell number (control antibody) −cell number (experimental antibody))/cell number (control antibody).Dose % T2 B Cell number/ % Antibody (μg/mouse) cells animal DepletionR347 10 21.3% 242,018 N/A R347 50 17.3% 166,575 N/A R347 250 23.0%212,106 N/A 3649 10 23.4% 95,554 60.5% 3649 50 18.9% 4,322 97.4% 3649250 16.6% 6,945 96.7% 3649-3M 10 26.4% 11,368 95.3% 3649-3M 50 10.9% 60799.6% 3649-TM 50 16.7% 135,944 18.4% 3649-TM 250 21.6% 93,662 55.8%

TABLE 11 Summary of splenic T3 B cell depletion results. 3649, 3649-3M,3649-TM, or control R347 antibody was administered to hCD19tg+/− micefollowing the protocol described above. T3 B cells are defined as theCD93+, IgM low, CD23+ fraction of transitional B cells (see FIG. 17B fordetails). ‘)/0 depletion was calculated as 100 × (cell number (controlantibody) − cell number (experimental antibody))/cell number (controlantibody). Negative depletion numbers are used when the size of a cellpopulation in the treated animal was larger than the corresponding sizein the control animal. Dose % T3 B Cell % Antibody (μg/mouse) cellsnumber/animal Depletion R347 10 34.3% 378,181 N/A R347 50 25.2% 242,767N/A R347 250 30.7% 306,514 N/A 3649 10 33.1% 135,097 64.3% 3649 50 25.4%5,652 97.7% 3649 250 21.1% 8,062 97.4% 3649-3M 10 26.2% 11,148 97.1%3649-3M 50 9.4% 518 99.8% 3649-TM 50 47.8% 399,685 (−64.6%) 3649-TM 25037.9% 172,129 43.8%

TABLE 12 Summary of splenic mature B cell depletion results. 3649,3649-3M, 3649-TM, or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. Mature B cellsare defined as the CD93− fraction of B cells (see FIG. 17B for details).% depletion was calculated as 100 × (cell number (control antibody) −cell number (experimental antibody))/cell number (control antibody).Negative depletion numbers are used when the size of a cell populationin the treated animal was larger than the corresponding size in thecontrol animal. % Dose mature B Cell number/ % Antibody (μg/mouse) cellsanimal Depletion R347 10 80.0% 5,175,585 N/A R347 50 80.7% 3,883,914 N/AR347 250 82.1% 4,454,167 N/A 3649 10 86.3% 3,042,745 41.2% 3649 50 96.2%794,477 79.5% 3649 250 94.3% 933,936 79.0% 3649-3M 10 88.7% 532,48289.7% 3649-3M 50 93.5% 108,806 97.2% 3649-TM 50 87.5% 5,724,882 (−47.4%)3649-TM 250 87.5% 3,792,089 14.9%

TABLE 13 Summary of splenic follicular B cell depletion results. 3649,3649-3M, 3649-TM, or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. Follicular Bcells are defined as the IgM+, CD23+ fraction of mature B cells (seeFIG. 17B for details). % depletion was calculated as 100 × (cell number(control antibody) − cell number (experimental antibody))/cell number(control antibody). Negative depletion numbers are used when the size ofa cell population in the treated animal was larger than thecorresponding size in the control animal. % Dose follicular Cell number/% Antibody (μg/mouse) B cells animal Depletion R347 10 78.2% 4,053,717N/A R347 50 69.3% 2,731,740 N/A R347 250 74.4% 3,298,335 N/A 3649 1076.9% 2,345,011 42.2% 3649 50 38.8% 310,160 88.6% 3649 250 45.6% 427,69187.0% 3649-3M 10 58.2% 306,833 92.4% 3649-3M 50 40.6% 40,611 98.5%3649-TM 50 79.9% 4,573,294 (−67.4%) 3649-TM 250 81.6% 3,091,310 6.3%

TABLE 14 Summary of splenic marginal zone B cell depletion results.3649, 3649-3M, 3649-TM, or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. Marginal zone Bcells are defined as the IgM high, CD23− fraction of mature B cells (seeFIG. 17B for details). % depletion was calculated as 100 × (cell number(control antibody) − cell number (experimental antibody))/cell number(control antibody). Negative depletion numbers are used when the size ofa cell population in the treated animal was larger than thecorresponding size in the control animal. % marginal Dose zone B Cellnumber/ % Antibody (μg/mouse) cells animal Depletion R347 10 10.6%546,769 N/A R347 50 14.3% 526,975 N/A R347 250 19.0% 861,171 N/A 3649 1010.8% 326,581 40.3% 3649 50 36.9% 291,707 44.6% 3649 250 40.1% 375,83456.4% 3649-3M 10 20.6% 110,411 79.8% 3649-3M 50 22.0% 26,845 94.9%3649-TM 50 14.6% 835,215 (−58.54%) 3649-TM 250 7.9% 297,329 65.5%

TABLE 15 Summary of bone marrow B cell depletion results. 3649, 3649-3M,3649-TM, or control R347 antibody was administered to hCD19tg+/− micefollowing the protocol described above. B cells are defined as theB220+, mouse CD19+ fraction of lymphocytes (see FIG. 17C for details).‘)/0 depletion was calculated as 100 × (cell number (control antibody) −cell number (experimental antibody))/cell number (control antibody).Dose % B Cell number/ % Antibody (μg/mouse) cells animal Depletion R34710 36.5% 1,423,555 N/A R347 50 23.0% 1,253,562 N/A R347 250 49.0%1,638,383 N/A 3649 10 23.9% 828,302 41.8% 3649 50 5.3% 192,629 84.6%3649 250 15.1% 325,810 80.1% 3649-3M 10 12.2% 380,642 73.3% 3649-3M 503.6% 100,271 92.0% 3649-TM 50 28.6% 1,192,432 4.9% 3649-TM 250 59.2%1,551,662 5.3%

TABLE 16 Summary of bone marrow pro-B cell depletion results. 3649,3649-3M, 3649-TM, or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. Pro-B cells aredefined as the CD43+, IgM− fraction of B cells (see FIG. 17C fordetails). % depletion was calculated as 100 × (cell number (controlantibody) − cell number (experimental antibody))/cell number (controlantibody). Negative depletion numbers are used when the size of a cellpopulation in the treated animal was larger than the corresponding sizein the control animal. Dose % pro-B Cell number/ Antibody (μg/mouse)cells animal % Depletion R347 10 8.0% 106,735 N/A R347 50 10.8% 135,709N/A R347 250 12.9% 298,552 N/A 3649 10 17.6% 143,882 (−34.8%) 3649 5044.6% 87,312 35.7% 3649 250 59.7% 233,214 21.9% 3649-3M 10 42.2% 160,572(−50.4%) 3649-3M 50 49.5% 50,159 63.0% 3649-TM 50 9.1% 110,932 18.3%3649-TM 250 18.9% 298,063 0.2%

TABLE 17 Summary of bone marrow pre-B cell depletion results. 3649,3649-3M, 3649-TM, or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. Pre-B cells aredefined as the CD43−, IgM− fraction of B cells (see FIG. 17C fordetails). % depletion was calculated as 100 × (cell number (controlantibody) − cell number (experimental antibody))/cell number (controlantibody). Dose % pre- Cell number/ % Antibody (μg/mouse) B cells animalDepletion R347 10 54.2% 786,256 N/A R347 50 53.4% 665,597 N/A R347 25044.9% 1,037,445 N/A 3649 10 44.3% 368,091 53.2% 3649 50 44.3% 82,85487.6% 3649 250 28.7% 112,154 89.2% 3649-3M 10 44.0% 166,139 78.9%3649-3M 50 43.4% 42,831 93.6% 3649-TM 50 52.5% 618,151 7.1% 3649-TM 25040.8% 631,574 39.1%

TABLE 18 Summary of bone marrow immature/mature-B cell depletionresults. 3649, 3649-3M, 3649-TM, or control R347 antibody wasadministered to hCD19tg+/− mice following the protocol described above.Immature/mature B cells are defined as the CD43−, IgM+ fraction of Bcells (see FIG. 17C for details). % depletion was calculated as 100 ×(cell number (control antibody) − cell number (experimentalantibody))/cell number (control antibody). Negative depletion numbersare used when the size of a cell population in the treated animal waslarger than the corresponding size in the control animal. % immature/Dose mature B Cell number/ % Antibody (μg/mouse) cells animal DepletionR347 10 34.7% 488,578 N/A R347 50 30.3% 382,746 N/A R347 250 36.1%835,327 N/A 3649 10 32.5% 267,869 45.2% 3649 50 7.8% 16,149 95.8% 3649250 7.4% 27,688 96.7% 3649-3M 10 9.9% 38,723 92.1% 3649-3M 50 4.3% 4,34098.9% 3649-TM 50 35.0% 421,214 (−10.1%) 3649-TM 250 34.6% 532,935 36.2%

TABLE 19 Summary of bone marrow immature B cell depletion results. 3649,3649-3M, 3649-TM, or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. Immature B cellsare defined as the CD93+ fraction of immature/mature B cells (see FIG.17C for details). % depletion was calculated as 100 × (cell number(control antibody) − cell number (experimental antibody))/cell number(control antibody). Negative depletion numbers are used when the size ofa cell population in the treated animal was larger than thecorresponding size in the control animal. Dose % B Cell number/ %Antibody (μg/mouse) cells animal Depletion R347 10 46.4% 230,643 N/AR347 50 47.6% 181,178 N/A R347 250 43.9% 183,209 N/A 3649 10 33.2%89,491 61.2% 3649 50 43.6% 7,542 95.8% 3649 250 18.2% 2,515 98.6%3649-3M 10 65.6% 24,592 89.3% 3649-3M 50 41.9% 1,780 99.0% 3649-TM 5039.4% 161,537 10.8% 3649-TM 250 37.1% 204,694 (−11.7%)

TABLE 30 Summary of bone marrow mature B cell depletion results. 3649,3649-3M, 3649-TM, or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. Mature B cellsare defined as the CD93 low/− fraction of immature/mature B cells (seeFIG. 17C for details). % depletion was calculated as 100 × (cell number(control antibody) − cell number (experimental antibody))/cell number(control antibody). Negative depletion numbers are used when the size ofa cell population in the treated animal was larger than thecorresponding size in the control animal. % Dose mature Cell number/ %Antibody (μg/mouse) B cells animal Depletion R347 10 52.0% 249,983 N/AR347 50 41.3% 159,933 N/A R347 250 49.2% 205,556 N/A 3649 10 63.6%169,712 32.1% 3649 50 22.6% 3,904 97.6% 3649 250 39.2% 5,435 97.4%3649-3M 10 31.4% 13,102 94.8% 3649-3M 50 18.7% 802 99.5% 3649-TM 5058.9% 252,755 (−58.0%) 3649-TM 250 55.8% 290,984 (−41.6%)

TABLE 31 Summary of peritoneal cavity B cell depletion results. 3649,3649-3M, 3649-TM, HB12B or control R347 antibody was administered tohCD19tg+/− mice following the protocol described above. Peritonealcavity B cells are defined as the IgM+ fraction of peritoneallymphocytes (see FIG. 17D for details). % depletion was calculated as100 × (% B cell (control antibody) − % B cell (experimental antibody))/%B cell (control antibody). Negative depletion numbers are used when thesize of a cell population in the treated animal was larger than thecorresponding size in the control animal. Dose Antibody (μg/mouse) % Bcell % Depletion R347 10 25.9% N/A R347 50 30.3% N/A R347 250 55.6% N/A3649 10 16.8% 35.3% 3649 50 20.1% 33.6% 3649 250 35.8% 35.6% 3649-3M 1015.3% 41.1% 3649-3M 50 13.1% 56.9% 3649-TM 50 26.7% 11.9% 3649-TM 25056.5% (−1.65%) HB12B 50 23.6% 22.0% HB12B 250 23.3% 58.2%

7.5.1. CD19 Density Influences the Effectiveness of CD19Antibody-Induced B Cell Depletion

To determine whether an anti-CD19 antibody's ability to deplete B cellsis dependent on CD19 density, anti-CD19 antibodies of the invention canbe administered to mice having varying levels of hCD19 expression. Theresults obtained will demonstrate whether human CD19 density on B cellsand antibody isotype can influence the depletion of B cells in thepresence of an anti-CD19 antibody. The same assay can be used todetermine whether other anti-CD19 antibodies can effectively deplete Bcells. The results can be correlated to treatment of human patients withvarying levels of CD19 expression. Thus, the methods for examining CD19presence and density, described herein, can be used in human subjects toidentify patients or patient populations for which certain anti-CD19antibodies can deplete B cells and/or to determine suitable dosages.

To determine whether CD19 density influences the effectiveness ofanti-CD19 antibody-induced B cell depletion representative blood andspleen B cell depletion can be examined in hCD19tg mice after treatmentwith the anti-CD19 antibodies of the invention (7 days, 250 μg/mouse).The results are expected to demonstrate that CD19 density influences theefficiency of B cell depletion by anti-CD19 antibodies in vivo. Forexample, low-level CD19 expression in hCD19tg mice would be expected tohave a marked influence on circulating or tissue B cell depletion by theadministered antibody. B cell clearance can be assessed 24 hours afteranti-CD19 or control mAb treatment of individual mice.

7.5.2. Determination Whether Tissue B Cell Depletion is FcγR-Dependent

Should administration of an anti-CD19 mAb of the invention result intissue B cell depletion, the following assays can be used to demonstratedependence upon FcγR expression. Through a process of interbreedinghCD19tg mice with mice lacking expression of certain FcγR, mice can begenerated that express hCD19 and lack expression of certain FcγR. Suchmice can be used in assays to assess the ability of anti-CD19 antibodiesto deplete B cells through pathways that involve FcγR expression, e.g.,ADCC. Thus, anti-CD19 antibodies identified in these assays can be usedto engineer chimeric, human or humanized anti-CD19 antibodies using thetechniques described above. Such antibodies can in turn be used in thecompositions and methods of the invention for the treatment of human Bcell disorders and diseases including, but not limited to, autoimmunediseases and disorders.

The innate immune system mediates B cell depletion following anti-CD20antibody treatment through FcγR-dependent processes. Mouse effectorcells express four different FcγR classes for IgG, the high-affinityFcγRI (CD64), and the low-affinity FcγRII (CD32), FcγRIII (CD16), andFcγRIV molecules. FcγRI, FcγRIII and FcγRIV are hetero-oligomericcomplexes in which the respective ligand-binding α chains associate witha common γ chain (FcRγ). FcRγ chain expression is required for FcγRassembly and for FcγR triggering of effector functions, includingphagocytosis by macrophages. Since FcRγ^(−/−) mice lack high-affinityFcγRI (CD64) and low-affinity FcγRIII (CD16) and FcγRIV molecules,FcRγ^(−/−) mice expressing hCD19 can be used to assess the role of FcγRin tissue B cell depletion following anti-CD19 antibody treatment.

7.5.3. Durability of Anti-CD19 Antibody-Induced B Cell Depletion

To assess the efficacy and duration of B cell depletion, hCD19tg micecan be administered a single low dose (e.g. 250 μg) injection ofanti-CD19 antibody and the duration and dose response of B celldepletion followed as a function of time. The results are expected todemonstrate that circulating B cells are depleted for a substantialamount of time (e.g. one week to six months), followed by a gradualrecovery of blood-borne B cells.

7.6. Persistence of CD19 on the Surface of B Cells after Administrationof Anti-CD19 Antibody

Whether CD19 internalization will influence B cell depletion in vivo canbe assessed by comparing cell-surface CD19 expression followingadministration of the anti-CD19 antibodies of the present invention. Forexample, cell surface CD19 expression and B cell clearance in hCD19tgmice treated with an anti-CD19 antibody of the present invention orisotype-matched control antibody (250 μg) in vivo can be studied as afunction of time. Thus, spleen B cells can be harvested and assayed forCD19 at time zero (prior to anti-CD19 administration), and at 1, 4, and24 hours post-antibody administration. Isolated B cells may also betreated in vitro with saturating concentrations of each anti-CD19antibody plus isotype-specific secondary antibody in vitro with flowcytometry analysis to visualize total cell surface CD19 expression.Where CD19 on the surface of B cells is maintained, it will indicatecontinued susceptibility to ADCC, CDC, and apoptosis. If CD19 persistson the cell surface following binding of an anti-CD19 antibody, the Bcell will remain accessible to the ADCC, CDC, or apoptotic activity.Such results would demonstrate, in part, why the anti-CD19 antibodiesand treatment regimens of the invention will be efficacious in providingtherapy for human B cell disorders and diseases including, but notlimited to, transplant rejection and autoimmune diseases and disorders.

7.7. Anti-CD19 Antibody Treatment May Abrogate Humoral Immunity andAutoimmunity

In the event CD19 therapy decreases B cell representation, then theassays described in this example can be used to demonstrate that theanti-CD19 antibodies of the invention are capable of eliminating orattenuating immune responses. These assays can also be used to identifyother anti-CD19 antibodies that can be used to engineer chimeric, humanor humanized anti-CD19 antibodies using the techniques described above.Such antibodies can in turn be used in the compositions and methods ofthe invention for the treatment of autoimmune diseases and disorders inhumans, as well as for transplantation therapy.

The effect of anti-CD19 antibody-induced B cell depletion on serumantibody levels can be assessed by giving hCD19tg mice a singleinjection of anti-CD19 antibody and then assessing the reduction inimmunoglobulin levels in those mice. For example, two-month-oldlittermates can be treated with a single injection of an anti-CD19antibody of the present invention or a control antibody (e.g. 250 μg) onday 0. Antibody levels are then determined by ELISA. It is expected thatthe results will show that after 1 to 2 weeks, serum IgM, IgG2b, IgG3,and IgA antibody levels are significantly reduced, and remain reducedfor at least 10 weeks.

The influence of B cell depletion on T cell-independent type 1 (T1-1)and type 2 (TI-2) antibody responses may also be assessed by immunizinghCD19tg mice with TNP-LPS or DNP-Ficol1 (at day zero), 7 days afteranti-CD19 antibody or control antibody treatment. Significanthapten-specific IgM, IgG, and IgA antibody responses are expected not tobe observed in anti-CD19 antibody-treated mice immunized with eitherantigen. Antibody responses to the T cell-dependent (TD) Ag, DNP-KLH,may also be assessed using mice treated with anti-CD19 antibody 7 daysbefore immunization, where it is expected that DNP-KLH immunized micetreated with anti-CD19 antibody will show reduced humoral immunity.

7.8. Anti-CD19 Antibody Treatment in Conjunction with Anti-CD22 AntibodyTreatment

The assay described herein can be used to determine whether combinationtherapies, e.g., anti-CD19 antibodies in combination with chemotherapy,toxin therapy or radiotherapy, have beneficial effects, such as anadditive or more that additive depletion in B cells. The results ofcombination therapies tested in animal models can be correlated tohumans by means well-known in the art.

Anti-CD20 antibodies are effective in depleting human and mouse B cellsin vivo. Therefore, the benefit of simultaneous treatment with ananti-CD19 antibody of the present invention and anti-CD20 (e.g.,MB20-11; see, Yazawa et al., Prot Natl Acad Sci USA. 102(42):15178-83(2005)) antibodies can be assessed to determine whether this willenhance B cell depletion. Mice can be treated with suboptimal doses(e.g. 2 μg, 5 μg, 10 μg, 20 μg, or 50 μg) of each antibody eitherindividually, or as a combination of both antibodies. It is expectedthat the results will demonstrate that simultaneous anti-CD19 andanti-CD20 antibody treatments are beneficial. In a similar manner, theefficacy may be determine for treatment with a combination of ananti-CD19 antibody of the present invention with an anti-CD22 antibody,or a combination of an anti-CD19 antibody of the present invention, ananti-CD20 antibody, and an anti-CD22 antibody.

7.9. Therapeutic Efficacy of Subcutaneous (S.C.) Administration of anAnti-CD19 Antibody of the Invention

The assay described herein can be used to determine whether asubcutaneous route of administration of an anti-CD19 antibody of theinvention can effectively deplete B cells. The results of the efficacyof different delivery routes tested in animal models can be correlatedto humans by means well-known in the art.

For example, hCD19tg mice can be treated with an anti-CD19 antibody ofthe invention at 250 μg either by subcutaneous (s.c.), intraperitoneal(i.p.) or intravenous (i.v.) administration. Values are determined forthe mean (±SEM) blood (per mL), bone marrow, spleen, lymph node, andperitoneal cavity B220⁺ B cell numbers on day seven as assessed usingflow cytometry. Results are expected to demonstrate that subcutaneous(s.c.), intraperitoneal (i.p.) and intravenous (i.v.) administration ofan anti-CD19 antibody of the invention will effectively depletecirculating and tissue B cells in vivo.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

7.10. Anti-CD19 Antibodies Reduce Tumor Growth in an In Vivo LymphomaModel

Anti-CD19 antibodies of the invention, which bind to human CD19, may beassessed for their ability to reduce tumor growth in in vivo animalmodels. For example, SCID mice would be injected with human lymphomacell lines to establish a tumor xenograft (e.g., s.c. injection of Rajicells). Subsequently, the mice would be given several doses of ananti-CD19 antibody of the invention (e.g., 100 lug antibody/mouse 5times). Tumor growth would be followed using standard methods (e.g.,tumor volume, animal weight, paralysis) The effect of anti-CD19treatment on tumor growth may be determined by comparing animalsreceiving anti-CD19 or control antibody treatment. The results obtainedusing anti-CD19 antibodies identified as capable of reducing tumorgrowth can be correlated to use in humans, and antibodies capable ofreducing tumor growth can be used in the compositions and methods of theinvention for the treatment of human B cell disorders and diseaseincluding, but not limited to, B cell malignancies.

To determine whether an anti-CD19 antibody's ability to reduce tumorgrowth is dependent on CD19 density, tumor cell lines with differentCD19 expression profiles may be tested in the above described in vivotumor growth assay. For example, Daudi, Raji, Namalwa, and Ramos cellsall have significant levels of CD19 on their cell surface; RPMI 8226multiple myeloma cell line on the other hand does not express CD19. Theresults obtained may demonstrate whether human CD19 density on the tumorcell surface can influence the tumor growth reducing activity of ananti-CD19 antibody. The results can be correlated to treatment of humanpatients with varying levels of CD19 expression. Thus, the methods forexamining CD19 presence and density, described herein, can be used inhuman subjects to identify patients or patient populations for whichcertain anti-CD19 antibodies can reduce the growth of malignant B cellsand/or to determine suitable dosages.

To determine whether an anti-CD19 antibody's ability to reduce tumorgrowth is dependent FcγR, the above described in vivo tumor growth assaywould be performed using SCID mice with compromised Fcγ receptoractivity (e.g., FcRγ^(−/−)). Through a process of interbreeding SOD micewith mice lacking expression of certain FcγR, SCID mice can be generatedthat also lack expression of certain FcγR (e.g., SCID, FcR γ^(−/−)mice). Such mice can be used in assays to assess the ability ofanti-CD19 antibodies to reduce tumor growth through pathways thatinvolve FcγR expression, e.g., ADCC. Based on the results, anti-CD19antibodies with increased ADCC can be engineered using the techniquesdescribed above. Such antibodies can in turn be used in the compositionsand methods of the invention for the treatment of human B cell disordersand diseases including, but not limited to, B cell malignancies.

Following are the details of experiments demonstrating the ability of3649 humanized anti-CD19 antibody to reduce tumor growth in an in vivoanimal model. On day 1 of the experiment, 4-6 weeks old female CB-17SCID mice are injected s.c. with 5×10⁶ Raji human lymphoma cells intheir hind flank. Raji cells express significant levels of CD19 on theirsurface and are sensitive to 3649 directed ADCC (see, FIGS. 7 and 8).Groups of 10 Raji cells injected animals are treated with a humanizedanti-CD19 antibody, control anti-CD20 antibody, isotype control antibodyof irrelevant specificity. Multiple different doses of an antibody maybe tested side by side. A non-limiting example for treatment schedule is5 biweekly i.p. doses of 10 mg/kg antibody starting on day 4. Anadditional control group of animals receiving PBS only is included aswell. Tumor volume is measured using standard methods. Animals withtumor volume greater than 2000 mm³ or with significant signs ofmorbidity are humanely euthanized. Tumor growth is plotted over time.

In the experiment summarized in FIG. 13, ten Raji cell injected animalseach were treated with 5 bi-weekly i.p. doses of 10 mg/kg (i) anti-CD20antibody, (ii) 3649-TM Fc variant with reduced ADCC, (iii) 3649antibody, or (iv) R347 control antibody starting on day 4. An additionalcontrol group of 10 animals received PBS only. Treatment with 3649anti-CD19 antibody and positive control anti-CD20 antibody significantlyreduced tumor growth. The standard deviation in tumor size for thegroups receiving 3649 or anti-CD20 antibody increased with time becauseof the presence of completely tumor free individuals in both treatmentgroups. The 3649-TM Fc variant did not have a significant effect ontumor growth, suggesting that the tumor growth reducing effect of 3649humanized anti-CD19 antibody is mediated through ADCC.

In the experiment summarized in FIG. 14, ten Raji cell injected animalseach were treated with 5 bi-weekly i.p. doses of (i) anti-CD20 antibodyat 10 mg/kg (anti-CD20), (ii) 3649-TM Fc variant with reduced ADCC at 10mg/kg (3649TM), (iii) 3649 antibody at 10 mg/kg (3649), (iv) 3649antibody at 2.5 mg/kg (3649*) or (v) R347 control antibody at 10 mg/kgstarting on day 4. Treatment with 3649 anti-CD19 antibody, 3649-3M humanADCC enhanced anti-CD-19 antibody and positive control anti-CD20antibody significantly reduced tumor growth. The 3649-TM Fc variantanti-CD19 antibody did not have a significant effect on tumor growth,suggesting that the tumor growth reducing effect of 3649 is mediatedthrough ADCC.

In the experiment summarized in FIG. 30, ten Raji cell injected animalseach were treated with 5 bi-weekly i.p. doses of (i) anti-CD20 antibodyat 10 mg/kg (anti-CD20), (ii) 3649 antibody at 10 mg/kg (3649), (iii)afucosylated 3649 antibody at 2.5 or 10 mg/kg (3649-aFuc) or (iv) R347control antibody at 10 mg/kg starting on day 4. Treatment with 3649anti-CD19 antibody, 3649-aFuc anti-CD-19 antibody and positive controlanti-CD20 antibody significantly reduced tumor growth.

7.11. Affinity Matured 3649 Anti-CD19 Antibodies

The following sections describe the identification of affinity maturedCDR variants of the 3649 anti-CD19 antibody that display an increasedbinding affinity towards cell surface displayed human CD19 antigencompared to the parental 3649 anti-CD19 antibody. The section alsodescribes the in vitro characterization of the affinity maturedanti-CD19 antibodies.

7.11.1. Identification of 3649 Variant Antibodies with IncreasedAffinity.

3649 variant anti-CD19 antibodies were identified by screening Fabfragment libraries comprising variant CDR sequences (see, US PatentApplication Publication No. US2006/0121042; Wu, H., Methods Mol Biol.,207:197-212 (2003); Wu & An, Methods Mol Biol., 207:213-33 (2003); Wu etal., J. Mol. Biol., 350:126-144 (2005); Wu et al., Proc. Natl. Acad.Sci. USA 95:6037-6042 (1998)).

Reagents: All chemicals were of analytical grade. Restriction enzymes,DNA-modifying enzymes, T4 ligase and T7 DNA polymerase were purchasedfrom New England Biolabs, Inc. (Beverly, Mass.). Custom oligonucleotideswere synthesized by Operon (Huntsville, Ala.). Streptavidin magneticbeads were purchased from Dynal (Lake Success, N.Y.).

Construction of 3649 Fab phage expression vector: Polynucleotidesencoding the VH and VK domains of 3649 anti-CD19 antibody were clonedinto an M13-based phage expression vector that facilitates theexpression of Fab fragments under the control of the lacZ promoter(Dall'Acqua et al., Methods 36:43-60 (2005)). The vector comprises thefirst constant region of the human γ1 heavy chain, the constant domainof a human kappa (κ) light chain and two annealing sites for the cloningof VH and VK genes. Cloning was carried out by hybridization mutagenesis(Kunkel et al., Proc. Natl. Acad. Sci. USA 82: 4778-82 (1985)) asdescribed in Wu & An, Methods Mol Biol., 207:213-33 (2003). Briefly,polynucleotides encoding the VH and VK region of 3649 were amplifiedusing approximately 0.5 pmol of each of the following gene-specificoligonucleotide primers: 3649 VH Fab Fw (SEQ ID NO:128), 3649 VH Fab Rev(SEQ ID NO:129), 3649 VK Fab Fw (SEQ ID NO:130), and 3649 VK Fab Rev(SEQ ID NO:131) (see Table 32). The 5′ nucleotide sequence of eachprimer comprises M13 vector specific sequences to allow for annealingbetween the PCR products and the single stranded vector. The nucleotidesequence of 3649 VH Fab Fw (SEQ ID NO:128) and 3649 VK Fab Fw (SEQ IDNO:130) primers comprises a 28/25 nucleotide sequence, respectively,corresponding to the M13 gene 3 leader sequence. The 5′ nucleotidesequence of 3649 VH Fab Rev (SEQ ID NO:129) and 3649 VK Fab Rev (SEQ IDNO:131) primers comprises a 28/30 nucleotide sequence corresponding tothe first constant region of the human γ1 heavy chain and the constantdomain of a human kappa (κ) light chain, respectively. Forward primers3649 VH Fab Fw (SEQ ID NO:128) and 3649 VK Fab Fw (SEQ ID NO:130) werebiotinylated to aid the isolation of the minus strand of the PCRfragments. Polynucleotides encoding the 3649 VH and VK genes were PCRamplified from the corresponding 3649 IgG expression constructsdescribed above using the 3649 VH Fab Fw (SEQ ID NO:128)/3649 VH Fab Rev(SEQ ID NO:129) and 3649 VK Fab Fw (SEQ ID NO:130)/3649 VK Fab Rev (SEQID NO:131) primer combinations, respectively. PCR products were purifiedby agarose gel electrophoresis/electroelution, and subsequentlyphosphorylated using T4 polynucleotide kinase (Roche). The minus strandof the PCR fragments was isolated by dissociating the double-strandedPCR product with sodium hydroxide, depleting the plus biotinylatedstrand using streptavidin-coated magnetic beads, and recovering theminus strands by ethanol precipitation. Equimolar amounts of theisolated minus strand of VH and VK PCR fragments were annealed to theuridinylated single strand M13 MD101-5A template, and treated with T4DNA polymerase (Roche), T4 DNA ligase (Roche) following themanufacturer's instructions. The M13 specific nucleotide sequencesincorporated into the isolated VH and VK minus strand polynucleotidesspecifically anneal them to two separate regions of the M13 vector DNA,each of which contain a palindromic loop comprising a recognition sitefor the Xba I restriction endonuclease. Because the palindromic loopsself anneal even in the presence of annealed VH or VK sequences, Xbaldigestion of the anncalcd, T4 DNA polymerase/T4 DNA ligase treated DNAcomplexes allows for the selection of newly synthesized minus vectorstrands comprising both VH and VK domains fused in frame with the humankappa κ constant and first human γ1 constant regions, respectively, atthe expense of the digested parental template plus strand. The reactionproduct was digested with Xbal, heat inactivated and electroporated intoDH1OB cells. Transformed DH1OB cells were titered on bacterial plateswith an Escherichia coli XL-1 Blue lawn. Phage DNA isolated from severalindependent plaques was sequenced to aid the identification of a cloneencoding the 3649 Fab fragment.

TABLE 32 PCR primers used to generate 3649 VH and VKencoding polynucleotides used forhybridization mutagenesis. 3649 specific residues are underlined. SEQName ID NO 3649 VH M13 Fw GCTGGTGGTGCCGTTCTATAGCCATAG 128CGAGGTGCAGCTGGTGGAGTCTGG 3649 VH M13 Rev GGAAGACCGATGGGCCCTTGGTGGAGG 129CTGAGGAGACGGTGACCAGGGTTCCTT G 3649 VK M13 Fw GGTCGTTCCATTTTACTCCCACTCCGA130 AATTGTGCTGACTCAGTCTCCAGACTT TCAG 3549 VK M13 RevGATGAAGACAGATGGTGCAGCCACAGT 131 ACGTTTGATCTCCACCTTGGTCCCTCC GCCGAACG

Generation of single CDR focused Fab libraries: Two separate singleamino acid substitution libraries were prepared for each of the six CDRregions of the 3649 antibody. The “NSS” libraries comprised all possiblesingle amino acid substituted variants of a given CDR wherein thesubstitute amino acid is any one of eight amino acids encoded by the NSSdegenerate codon. The “NWS” libraries comprise all possible single aminoacid substituted variants of a given CDR wherein the substitute aminoacid is any one of twelve amino acids encoded by the NWS degeneratecodon. Individual libraries were prepared by hybridization mutagenesisof the 3649 Fab encoding phage vector described above (Kunkel et al.,Proc. Natl. Acad. Sci. USA 82: 4778-82 (1985); Wu & An, Methods MolBiol., 207:213-33 (2003)). Briefly, two sets of degenerate minus strandoligonucleotides were prepared for each of the six CDRs. The “NSS” and“NWS” sets comprised all possible single codon substitutions of a CDRregion with the NSS and NWS, respectively, degenerate codon. Theoligonucleotide sets used for the preparation of the heavy chain CDR1focused libraries are listed in Table 33 as an example. Each degenerateminus strand oligonucleotide was phosphorylated prior to annealing at a10:1 molar ratio to a uridinylated single stranded 3649 Fab phagetemplate DNA. The temperature of the annealing reaction was lowered from95° C. to 55° C. over 1 hour. T4 ligasc and T7 DNA polymerase was addedto the annealed material and incubated for 1.5 hours at 37° C. The finalnegative strand synthesis products obtained using differentoligonucleotides from a single CDR specific set were pooled; the NSS andNWS pools, however, were maintained separately and screenedindependently. Typically, one pl of the pooled negative strand synthesisreaction was electroporated into XL1-Blue for plaque formation onXL1-Blue bacterial lawn. Individual phage clones are eluted 200 pl of 10mM Tris (pH 7.4), 100 mM NaCl buffer and stored at 4° C. The library maybe characterized by sequencing 24 randomly selected phage clones todetermine the distribution of mutations within the CDR as well as tocalculate the mutagenesis rate.

TABLE 33 Oligonucleotides used for the generation of3649 heavy chain CDR1 focused libraries. TheNSS and NWS codon comprising oligonucleotideswere used for the generation of the separate “NSS” and “NWS”libraries. Nucleotides encoding CDR residues are underlined. SEQ NameID NO HCDR1/ GCCTGGCGGACCCASSNCATCCAAGAGCTACTGAA 132 NSS1 GGTGAATCCAGHCDR1/ GCCTGGCGGACCCAGTTSSNCCAAGAGCTACTGAA 133 NSS2 GGTGAATCCAG HCDR1/GCCTGGCGGACCCAGTTCATSSNAGAGCTACTGAA 134 NSS3 GGTGAATCCAG HCDR1/GCCTGGCGGACCCAGTTCATCCASSNGCTACTGAA 135 NSS4 GGTGAATCCAG HCDR1/GCCTGGCGGACCCAGTTCATCCAAGASSNACTGAA 136 NSS5 GGTGAATCCAG HCDR1/GCCTGGCGGACCCAWSNCATCCAAGAGCTACTGAA 137 NWS1 GGTGAATCCAG HCDR1/GCCTGGCGGACCCAGTTWSNCCAAGAGCTACTGAA 138 NWS2 GGTGAATCCAG HCDR1/GCCTGGCGGACCCAGTTCATWSNAGAGCTACTGAA 139 NWS3 GGTGAATCCAG HCDR1/GCCTGGCGGACCCAGTTCATCCAWSNGCTACTGAA 140 NWS4 GGTGAATCCAG HCDR1/GCCTGGCGGACCCAGTTCATCCAAGAWSNACTGAA 141 NWS5 GGTGAATCCAG

Primary screen of the single CDR focused Fab libraries: The primaryscreen consisted of a single point ELISA (SPE) performed using thesecreted Fab containing supernatant of 1 ml phage cultures andrecombinant human CD19 expressing 300B4 cells as a capture agent. Anexhaustive screen of a library can usually be achieved by testing anumber of individual clones that equals three times the size of thelibrary taking into account the rate of mutagenesis. For example,exhaustion of a VH CDR1 (5 amino acid residues) “NSS” substitutionlibrary (8 possible amino acids encoded by the degenerate NSS codon)with a 50% mutagenesis rate may be achieved by screening 5×8×2=80randomly selected individual clones. In the experiment described herein˜400 clones were screened, however, for each library regardless of theCDR length, or synthesis efficiency. Supernatant of small scale phagecultures were isolated as described in Wu & An, Methods Mol Biol.,207:213-33 (2003). Briefly, 0.75 mL of exponentially growing TG1 cellsare inoculated with 75 μl of eluted phage stock in the presence of 0.5mM IPTG and incubated at 37° C. for 1 hr in 96-well plates. The platecultures are moved to room temperature and grown overnight on an orbitalshaker. Bacteria from 0.36 ml of the overnight cultures is collected byfiltration through a low protein binding nylon membrane (e.g., Silentscreen plate from Nalgene) and treated on the filter with 200 μl of TESbuffer (30 mM Tris pH 8.0, 2 mM EDTA, 20% sucrose) with 2 mg/ml lysozymefor 10 minutes at room temperature to release the secreted Fab from theperiplasmic space. Fab fragment containing extracts are collected byfiltration. Fab fragment concentration in the extract is determinedusing standard assays. 300B4 cell based ELISA was performed as describedsupra. The parental 3649 Fab expressing phage clone was included in thecell based ELISA assays as a positive control. Relative binding affinityof variant Fab fragments was compared by plotting the ELISA signalagainst Fab concentration. For example, FIG. 18 shows the resultsobtained with 3649 variant Fabs comprising a random single amino acidsubstitution within VH CDR3. Fabs 4B7 and 4G6 display significantlyincreased binding affinity to human CD19 expressing 300B4 cells comparedto that of the parental 3649 Fab.

Secondary screen of the single CDR focused Fab libraries: CDR variantFab clones with a primary screen ELISA signal of at least 10% higherthan that of the 3649 parental Fab were re-grown at a 15 ml scale, andre-assayed by the same 300B4 cell based ELISA in duplicate wells toconfirm the positive result. 15 ml scale Fab extract were preparedfollowing the protocol described in Wu, H., Methods Mol Biol.,207:197-212 (2003). Fab concentration was determined using standardassays. 300B4 cell based ELISA was performed as described supra. The3649 parental Fab was included in the assay as a positive control.Clones with a secondary screen signal of at least 10% higher than thatof the 3649 control Fab were sequenced to determine the identity ofsingle amino acid substitutions leading to increased human CD19 antigenbinding. Amino acid substitutions identified by sequencing the isolatedaffinity improved anti-CD19 Fab clones are listed in Table 34.

TABLE 34 List of beneficial single amino acid substitutions isolatedfrom the single CDR focused Fab libraries. Amino acid positions arenumbered according to Kabat. Substi- Posi- Substi- Posi- Clone tutiontion Clone tution tion VH 2A11 Ser > Thr 32 VL 8A10 Asp > Ile   27C CDR11H1 Trp > Glu 33 CDR1 9D11 Thr > His   27D 5H12 Trp > Leu 33 2B8 Ile >Leu 30 1A8 Asn > Phe 35 1C10 Met > Arg 33 1C5 Asn > Tyr 35 1H10 Met >Thr 33 3C3 Asn > Asp 35 12E3 Met > Ile 33 6H6 Asn > Leu 35 VH 3A11 Thr >Ser 57 VL 10D3 Ala > Tyr 50 CDR2 4G8 Thr > Pro 57 CDR2 10G4 Ala > Glu 503E9 Thr > Asn 57 2C12 Gln > Thr 54 4D4 Asn > Leu 60 4F10 Gln > Pro 545A11 Asn > Tyr 60 9B10 Gly > Tyr 55 2H11 Gly > Ala 61 VH 4B7 Leu > Arg 100B VL 2F7 Ser > Thr 91 CDR3 7E3 Leu > His  100B CDR3 6E10 Phe > Ile96 12E3 Leu > Phe  100B 2C12 Phe > Asn 96 7H11 Leu > Tyr  100B 11G12Thr > Pro 99

Generation of combinatorial Fab library: Two combinatorial librariescomprising all possible combinations of beneficial single amino acidsubstitutions identified from the CDR focused libraries were generatedby hybridization mutagenesis. The first combinatorial library wasgenerated using a set of degenerate oligonucleotides that encoded boththe parental 3649 residues as well as the identified beneficial singleamino acid substitutions (Table 35). A second combinatorial library wasgenerated using a separate set of degenerate oligonucleotides thatencodes only the most beneficial single amino acid substitution residuesbut not the 3649 parental residues (Table 36). The two libraries weregenerated and screened separately. Library generation was done asdescribed in Wu, H., Methods Mol Biol., 207:197-212 (2003); Wu & An,Methods Mol Biol., 207:213-33 (2003). Briefly, degenerate primer setswere synthesized and phosphorylated. Hybridization mutagenesis wasperformed by using all primers in a single annealing and synthesisreaction. Library generation, testing and screening was performed asdescribed supra. The ELISA profile of the six combinatorial Fab cloneswith the highest binding affinity for human recombinant CD19 expressing300B4 cells is shown in FIG. 19. Clone 7E12 was recovered from the firstcombinatorial library generated with degenerate oligonucleotidesencoding both the parental and beneficial CDR substitution residues.Clones 14H5, 15D7, 15D1, 16C9, and 16C4 were recovered from the secondcombinatorial library generated with degenerate oligonucleotidesencoding only the most beneficial CDR substitution residues. All phageclones listed in FIG. 19 were sequenced to determine the amino acidsequence of the variant CDR regions with increased human CD19 bindingaffinity.

TABLE 35 Degenerate oligonucleotides for combinatorialphage library generation. The oligonucleotideset listed herein encodes both the parentalresidues and the beneficial substitutionresidues identified from the CDR focused single substitution libraries.SEQ Name ID NO. H1CDR1H CTGGCGGACCCAGTHCATCMAAGW 142 GCTACTGAAGGTGAATCCH2CDR1HP3E CTGGCGGACCCAGTHCATCTCAGW 143 GCTACTGAAGGTGAATCC H3CDR1HP3WP4LCTGGCGGACCCAGAGCATCMAAGW 144 GCTACTGAAGGTGAATCC H4CDR1HP3EP4LCTGGCGGACCCAGAGCATCTCAGW 145 GCTACTGAAGGTGAATCC H5CDR2HCTGCCCTTGAACTTCSCATWGTAG 146 TTAGDATCTCCATCTCCAG H6CDR2HP9NCTGCCCTTGAACTTCSCATWGTAG 147 TTATTATCTCCATCTCCAG H7CDR2HP12LCTGCCCTTGAACTTCSCAAGGTAG 148 TTAGDATCTCCATCTCCAG H8CHR2HP9N,CTGCCCTTGAACTTCSCAAGGTAG 149 P12L TTATTATCTCCATCTCCAG H9CDR3HP8H/YCCCAGTAGTCAAAGTCGTRAACCG 150 TAGKAATAAATCCTGATCTAGC H10CDR3HP8FCCCAGTAGTCAAAGTCSAAACCGT 151 AGKAATAAATCCTGATCTAGC H11CDR3HP8RCCCAGTAGTCAAAGTCTCGAACCG 152 TAGKAATAAATCCTGATCTAGC L12aCDR1Lp71p8HCTGCTGGAACCAGTTYVTAAAACT 153 AAKGCCAAAATGAATAACACTTTC GCTGGCL13aCDR1Lp8H CTGCTGGAACCAGTTYVTAAAACT 154 AAKGCCAAAATGATCAACACTTTCGCTGGC L14aCDR1Lp71 CTGCTGGAACCAGTTYVTAAAACT 155AAKGCCAAAAGTAATAACACTTTC GCTGGC L15aCDR1Lp11p14 CTGCTGGAACCAGTTYVTAAAACT156 AAKGCCAAAAGTATCAACACTTTC GCTGGC L16CDR2Lp1Y GGGGACCCCGGATCCTTGATTGGA157 TGCATAATGGATGAGGAGCTTTGG L17CDR2Lp1Yp6Y GGGGACCCCGGAGTATTGATTGGA 158TGCATAATGGATGAGGAGCTTTGG L18CDR2Lp1Yp5T/P GGGGACCCCGGATCCTGKATTGGA 159TGCATAATGGATGAGGAGCTTTGG L19CDR2Lp1E/A GGGGACCCCGGATCCTTGATTGGA 160TGCCKCATGGATGAGGAGCTTTGG L20CDR2Lp1E/Ap6Y GGGGACCCCGGAGTATTGATTGGA 161TGCCKCATGGATGAGGAGCTTTGG L21CDR2Lp1E/Ap5T/P GGGGACCCCGGATCCTGKATTGGA 162TGCCKCATGGATGAGGAGCTTTGG L22CDR3Lp3T/Sp8F/T CTCCGCCGAACGTGAWTGGAACCT 163CCTTAGWTTGCTGACAGTAATACG L23CDR3Lp3T/Sp8N CTCCGCCGAACGTGTTTGGAACCT 164CCTTAGWTTGCTGACAGTAATACG

TABLE 36 Degenerate oligonucleotides forcombinatorial phage library generation.The oligonucleotide set listed herein encodesonly the most beneficial substitutionresidues identified from the CDR focused single substitution libraries.SEQ Name ID NO. L24CDR1Lp7Ionly AACTAATGCCAAAaGTAatAAC 165ACTTTCGCTGGCTCTG L25CDR1Lp7Ip9Honly CATAAAACTAATGCCAAAatgA 166atAACACTTTCGCTGGCTCTGC AGG L26CDR1Lp8Honly CCAGTTCATAAAACTAATGCCA 167AAatgATCAACACTTTCGCTGG CTC L27CDR2Lp1Eonly GGATCCTTGATTGGATGCctCA 168TGGATGAGGAGCTTTGG L28CDR3Lp8I/Nonly GTCCCTCCGCCGAACGTGwtTG 169GAACCTCCTTACTTTGC H29CDR1Hp3Eonly CCTGGCGGACCCAGTTCATCtc 170AGAGCTACTGAAGGTGAATCC H30CDR1Hp3Ep5Yonly CCTGGCGGACCCAGTaCATCtc 171AGAGCTACTGAAGGTGAATCC H31CDR1Hp5Yonly CCTGGCGGACCCAGTaCATCCA 172AGAGCTACTGAAGGTGAATCC H32CDR2Hp13Aonly GAATCTGCCCTTGAACTTCgCA 173TTGTAGTTAGTATCTCCATC H33CDR3Hp8Ronly CTTGGCCCCAGTAGTCAAAGTC 174cgAACCGTAGTAATAAATCCTG H34CDR3Hp8R/Honly CTTGGCCCCAGTAGTCAAAGTC 175gygAACCGTAGTAATAAATCCT G

7.11.2. Characterization of Increased Affinity Variant Anti-CD19Antibodies

Polynucleotides encoding the variable regions of 7E12, 14H5, 15D7, 15D1,16C9, and 16C4 Fab variants with improved anti-CD19 binding activitywere PCR-amplified from the corresponding V region-encoding M13 phagevectors using pfu DNA polymerase (see, Dall'Acqua et al., Methods36:43-60 (2005)). The polynucleotides were then individually cloned intomammalian expression vectors encoding a human cytomegalovirus majorimmediate early (hCMVie) enhancer, promoter and 5′-untranslated region(M. Boshart, et al., Cell 41:521-530 (1985)). In this system, a human γ1chain is secreted along with a human is chain (S. Johnson, et al.,Infect. Dis. 176:1215-1224 (1997)). The different constructs wereexpressed transiently in HEK-293 cells and harvested 72 and 144 hourspost-transfection. The secreted, soluble human IgG1s were purified fromthe conditioned media directly using 1 ml HiTrap protein A columnsaccording to the manufacturer's instructions (APBiotech, Inc.,Piscataway, N.J.). Purified human IgG1s (typically >95% homogeneity, asjudged by SDS-PAGE) were dialyzed against phosphate buffered saline(PBS), flash frozen and stored at −70° C.

7.11.2.1. Cell Based ELISA Assays

The ability of 7E12, 14H5, 15D7, 15D1, 16C9, and 16C4 IgG antibodies tobind human CD19 was assessed in a cell based CD19 binding assays. Threedifferent cell lines were used as capture reagents: (i) humanrecombinant CD19 expressing 300B4 cells (FIG. 20.), (ii) Raji cells(FIG. 21.), and (iii) Daudi cells (FIG. 22.). All three cell lines werecultured according to standard protocols. A standard ELISA procedure canbe used for the cell based CD19 binding assay. For example, individualwells of a 96-well U bottom plate are seeded with 1×10e5 300B4 cells andincubated overnight. Cells are washed once with ELISA buffer prior toincubation on ice with various amounts of anti-CD19 antibodies. Bindingreactions are performed in triplicates for each antibody concentrationtested. Positive control wells using 3649 anti-CD19 antibody areincluded in the assay. Following incubation with the antibody, 300B4cells are washed three times with 200 micro liter of ELISA buffer. Cellsurface bound anti-CD19 antibodies are detected using a goat anti-humankappa antibody conjugated with horseradish peroxidase according tostandard protocols.

ELISA binding curves of 3649, 7E12, 14H5, 15D7, 15D1, 16C9, and 16C4anti-CD19 IgG antibodies using 300B4 cells, Raji cells, or Daudi cellsas capture reagents are shown in FIGS. 20-22. All of the testedantibodies except for 16C9 and 15D1 display significantly higher bindingaffinity for cell surface displayed human CD19 than the control 3649antibodies. The binding affinity of 16C9 and 15D1 matches that of the3649 antibody when 300B4 cells are used as capture reagent. The bindingaffinity of 16C9 and 15D1 antibodies to human CD19 is higher than thatof the control 3649 antibody when Raji cells or Daudi cells are used ascapture reagents.

7.11.2.2. 14H5 Anti-CD19 Antibody Variants with Modified DeamidationSite.

The primary amino acid sequence of the 3649, 7E12, 14H5, 15D7, and 16C9antibodies comprises an NG (residues 60-61 of VH CDR2) deamidationmotif. Three deamidation minus variants of 14H5 were generated bychanging the asparagine (N) residue at Kabat position 60 to tyrosine(Y), aspartic acid (D), or leucine (L). The Y60, D60, and L60 comprising14H5 variant VH regions are designated as 14H5-YG (SEQ ID NO:107),14H5-DG (SEQ ID NO:108), and 14H5-LG (SEQ ID NO:109), respectively.Antibody expression vectors comprising polynucleotides encoding thedeamidation minus 14H5 variants were generated using standard molecularcloning techniques. Transiently expressed 14H5-YG, 14H5-DG, and 14H5-LGanti-CD19 IgG was purified as described above. The binding affinity of14H5-YG, 14H5-DG, and 14H5-LG antibodies was ascertained using a cellbased ELISA assay utilizing the human recombinant CD10 expressing 300B4cells as a capture reagent. The 14H5 and 16C4 anti-CD10 antibodies wereemployed as positive controls. Results obtained are presented in FIG.23. The binding affinity of 14H5-YG, 14H5-DG, and 14H5-LG antibodies torecombinant human CD19 expressing 300B4 cells is lower than that ofeither the 14H5 or 16C4 antibodies.

7.11.2.3. Kinetic Off Rate of Affinity Matured Anti-CD19 Antibodies.

The kinetic off rate of 7E12, 14H5, 15D7, 15D1, 16C9, and 16C4 anti-CD19IgG antibodies was ascertained by following over time the elimination ofcell surface bound anti-CD19 antibodies. Briefly, Ramos cells wereincubated with the 7E12, 14H5, 15D7, 15D1, 16C9, or 16C4 affinitymatured anti-CD19 antibody following standard staining protocols. Cellswere washed following the incubation to eliminate any unbound primaryantibody and further incubated at 37° C. for 0, 30, or 60 minutes. Atthe end of the incubation period, cells were stained with an RPEconjugated mouse anti-human IgG Fc fragment secondary reagent followingstandard protocols and analyzed on a flow cytometer. Control batches ofcells were incubated with the 3649 anti-CD19 antibody or a referencecontrol anti-CD20 antibody prior to incubation at 37° C. Meanfluorescence intensity measured at various time points using thedifferent antibodies is displayed in FIG. 24A. 100% mean fluorescenceintensity corresponds to the staining intensity seen at time 0 with agiven antibody.

The loss of mean fluorescence intensity observed with the affinitymatured 7E12, 14H5, 15D7, 15D1, 16C9, and 16C4 anti-CD19 antibodies islower than that of observed with the anti-CD20 reference controlantibody. In contrast, the 3649 anti-CD19 antibody stained Ramos cellsdisplay a faster loss of mean fluorescence intensity that cells stainedwith the reference control anti-CD20 antibody (FIG. 24A).

In a separate experiment, Ramos cells were stained with an Alexa 647conjugated 16C4, 3649, or HB12B anti-CD19 antibody following standardprotocols. Following the staining, cells were washed to remove unboundantibody and further incubated at 37° C. for 0, 30, or 60 minutes. Cellswere subsequently analyzed on a flow cytometer. A fluorescentlyconjugated anti-CD20 antibody was included in the experiment as areference control. Mean fluorescence intensity (MFI) seen at varioustime points is shown in FIG. 24B. MFI is expressed as a fraction of theMFI value seen at time 0. Loss of MFI detected with the 16C4 affinitymatured anti-CD19 antibody is significantly slower than the loss of MFIseen with the 3649 or HB12B anti-CD19 antibodies. The anti-CD20reference control antibody displays the same off rate as the 3649 andHB12B anti-CD19 antibodies.

7.11.2.4. Cell Surface Staining by Affinity Matured Anti-CD19 Antibodies

Daudi cells were immunostained with 7E12, 14H5, 15D7, 15D1, 16C9, and16C4 anti-CD19 antibodies and a RPE conjugated goat anti-human IgG(Fab′)2 fragment secondary reagent according to standard protocols.Immunostained cells were analyzed on a flow cytometer. Medianfluorescence intensity (MFI) of stained cells at various primaryantibody concentrations is plotted in FIG. 25. Cells stained with the3649 anti-CD19 antibody were included as a reference control. Stainingintensity detected with 7E12, 14H5, 15D7, 16C9, and 16C4 anti-CD19antibodies was higher than that of the 3649 antibody stained cells atall concentrations tested. Staining intensity detected with 15D1 wassimilar to the staining intensity detected with the 3649 antibody at lowantibody concentrations (0.5 mg/ml or lower). MFI of 15D1 stained cellswas higher, however, than that of the 3649 antibody stained cells atelevated antibody concentrations (1 mg/ml or higher).

7.11.2.5. In Vitro ADCC Activity of Affinity Matured Anti-CD19Antibodies.

In vitro ADCC activity of affinity matured anti-CD19 antibodies wasmeasured using assays described herein. For example, results obtainedwith 16C4, 14H5, and 14H5-DG antibodies using Daudi target cells arepresented in FIG. 26. The 3549 anti-CD19 antibody was used as areference control. All three affinity matured antibody displayedincreased ADCC activity at antibody concentrations of 0.1 mg/ml or lesscompared to the 3649 reference control antibody. The ADCC activity of16C4, 14H5, and 14H5-DG antibodies matched the activity of the 3649antibody at 1 mg/ml or higher antibody concentrations.

In vitro ADCC activity of affinity matured afucosylated anti-CD19antibodies was also measured using assays described herein. For example,afucosylated 16C4 antibody (16C4-aFuc) mediated ADCC was measured usingDaudi target cells (FIG. 26). The experiments also included 16C4,3649-aFuc, and anti-CD20 antibodies as reference controls. ADCC of16C4-aFuc is significantly higher than that of the 3649-aFuc, anti-CD20,or fucosylated 16C4 reference antibodies. ADCC mediated by 16C4-aFuc iscomparable to that of the antibodies.

In vitro ADCC activity of 16C4, 16C4-aFuc, and 3649-aFuc anti-CD19antibodies were further characterized in a standardized in vitro ADCCassay using a variety of target cells. An anti-CD20 antibody wasincluded in the assay as a control. Target cells used represent avariety of B cell malignancies as well as different CD19 cell surfacedensities (Table 37). Relative surface expression of CD19 and CD20 ofthe target cells was determined by flow cytometry following standardprotocols. Table 37 lists the mean fluorescence intensity (MFI) oftarget cells stained with fluorescently labeled anti-CD20 or 16C4anti-CD19 antibodies. ADCC reactions were performed following theprotocols described above. Reactions were set up in triplicates using50,000 effector cells and 20,000 target cells to achieve an E:T ratio of2.5:1. Transgenic NK cells expressing CD16 and associated signalingpolypeptide FCERI-y served as effector cells. ADCC reactions wereallowed to proceed for 4 hours at 37° C. ADCC activity was determined atvarious antibody concentrations. Data was plotted as % cytotoxicity as afunction of antibody concentration. Maximum cell killing and EC50 values(antibody concentrations required for half-maximum cytotoxicity underthe conditions used) were established for target cell/antibodycombinations using standard methods. Table 37 presents the finalresults. Oci-LY19, KArpas-422, Nalm-6, and Namalwa cells representingDLCL, NHL, ALL, and Burkitt's lymphoma, respectively, were sensitive to16C4-aFuc antibodymediated cytotoxicity but largely not sensitive toanti-CD20 mediated ADCC. Daudi, Toledo, RL and Raji cells weresignificantly more sensitive to 16C4-aFuc antibody medited cytotoxicitythan to anti-CD20 mediated ADCC.

TABLE 37 Quantitative assessment of 16C4, 16C4-aFuc, and 3649-aFucanti-CD19 antibody mediated ADCC. MFI@1 μg/ml: mean uorescence intensityof cells stained with 1 μg/ml fluorescently labeled antibody; ADCC EC50:antibody concentration yielding half maximum killing under theconditions used; % max. cell kill at 10 μg/ml: maximum target cellkilling achived with 10 μg/ml antibody in the ADCC assay under theconditions used anti-CD20 16C4-aFuc 3649-aFuc 16C4 anti-CD20 16C4 ADCC %max. ADCC % max. ADCC % max. ADCC % max. MFI @ MFI @ EC50 cell kill EC50cell kill EC50 cell kill EC50 cell kill 1 ug/ml 1 ug/ml [ng/ml] at 10ug/ml [ng/ml] at 10 ug/ml [ng/ml] at 10 ug/ml [ng/ml] at 10 ug/ml TargetDaudi (Burkitt) 253 161 0.041  51 0.0031 50 0.0576 51 0.0576 16 cellGranta-519 403 75 0.0663 34 n.d. 6 n.d. 9 n.d. 0 lines (NHL/MCL) Toledo(DLCL) 312 472 0.0319 55 0.0075 48 0.0155 53 0.0545 12 Oci-LY19 (DLCL)12 258 n.d. 4 0.0163 47 0.0518 54 n.d. 6 Karpas-422 (NHL) 45 53 n.d. 50.0181 62 0.0442 64 n.d. 6 Farage (DLCL) 581 226 0.0113 54 0.0141 480.0536 48 0.0927 13 Nalm-6 (ALL) 26 448 n.d. 14 0.0199 35 0.0605 39 n.d.17 Karpas-1106P 102 66 0.0137 71 0.009  59 0.0142 59 0.533  16 DB (DLCL)70 61 0.2399 46 n.d. 34 n.d. 42 n.d. 6 RL (NHL) 261 344 0.0296 56 0.007345 0.0194 46 0.0393 15 Raji (Burkitt) 422 823 0.008  40 0.003  43 0.009 44 0.012  26 Namalwa (Burkitt) 664 298 n.d. 7 0.012  23 0.04  25 n.d. 4

7.11.2.6. In Vivo B Cell Depletion by Affinity Matured Anti-CD19Antibodies

Affinity matured anti-CD19 antibodies were tested in a B cell depletionassay essentially as described above. C57B16 hCD19 tg+/− animals weretreated with a single i.v. dose of 10, 50, or 250 μg of 16C4 affinitymatured anti-CD19 antibody (16C4) or 14H5DG affinity matured anti-CD19antibody (14H5DG). Reference control animals were treated with (i) 3649anti-CD19 antibody (3649), (ii) ADCC enhanced Fc variant of 3649anti-CD19 antibody (3649 3M), and (iii) afucosylated 3649 anti-CD19antibody (3649-aFuc). Negative control animals were treated with (i) theADCC compromised Fc variant of 3649 anti-CD19 antibody (3649 TM) or (ii)an antibody of irrelevant specificity (R347). Circulating lymphocytesand splenic lymphocytes were isolated 7 days after antibody treatment.Isolated cells were immunostained as described in Table 5 to identifyvarious B cell populations. Samples were ananlysed on a flow cytometerfollowing standard protocols.

The 16C4 affinity matured anti-CD19 antibody achieved a slightly higherdepletion of B cells than the 3649 anti-CD19 parent antibody. The3649-aFuc and 3649 3M antibodies achieved better depletion than the 16C4affinity matured antibody. The 14H5DG affinity matured anti-CD19antibody is less efficient at depleting B cells than the 3649 anti-CD19parent antibody.

7.11.2.7. Long Term Recovery of B Lymphocytes Following Administrationof a Single Depleting Dose of Affinity Matured Anti-CD19 Antibodies.

Long term recovery of the B cell compartment was studied following theadministration of a single depleting dose of 16C4-aFuc anti-CD19monoclonal antibody. Twenty five C57B16 hCD19 tg+/− mice (13 males, 12females, 2.5-3 months of age) were divided into 5 groups. A week beforeadministering the experimental antibodies (week −1) the animals wereexamined for overall health, weighed and a small aliquot of blood wascollected for each one of them for analysis. On day 0 of the experiment250 μg of 16C4-aFuc mAb, 50 μg of 16C4-aFuc mAb, 10 μg of 16C4-aFuc mAb,250 mg of R347 control antibody of irrelevant specificity, or PBS wereadministered intravenously to animals in groups 1, 2, 3, 4, and 5,respectively. On day 7 (week 1), and at weekly intervals afterwards,mice in each group were examined and bled. Blood samples were subjectedto flow cytometry to determine B cell, T cell, NK T cell, neutrophil,monocyte and dendritic cell numbers. The blood samples were furtheranalysed to determine the serum concentration of IgM, IgG1, IgG2b,IgG2c, IgG3, IgA, anti-dsDNA IgM, anti-dsDNA IgG, anti-ssDNA IgM,anti-ssDNA IgG as well as the serum levels of IL-7, CXCL12, CXCL13 andBAFF. Measurements were performed following standard procedures. Theoutline of the experiment and results obtained are presented in FIG. 38.

No obvious adverse effect was observed following drug treatment. Animalsin all experimental groups maintained normal activity levels and weight(FIG. 38B). B cell levels of animals receiving 16C4-aFuc anti-CD19antibody was significantly lower than that of the control animals (FIGS.38C and D). B cell depletion was complete even in animals receiving 10mg of 16C4-aFuc. The duration of B cell depletion was dose dependent;the duration of depletion increased with higher dose of 16C4-aFucantibody. B cells on animals receiving 10 μg 16C4-aFuc started torecover by week 3 and reached normal levels by week 5. Recovery of theanimals receiving 50 μg of 16C4-aFuc took 9 weeks. Animals receiving 250mg of 16C4-aFuc were still almost completely without B cells at week 11of the experiment. Blood concentration of T cells, NK-T cells, NK cells,dendritic cells, neutrophils, and monocytes were not affected by the16C4-aFuc antibody (data not shown). Serum levels of IgM, IgG1 and IgG2bwere also reduced by 16C4-aFuc antibody treatment (FIGS. 38E-G).Reduction in immunoglobulin levels was dose sensitive; significantreduction in levels was only seen in animals treated with 50 or 250 μg16C4-aFuc antibody. Recovery in immunoglobulin levels largely trackrecovery of the B cell compartment.

7.11.2.8. TEF-PAGE Analysis of Anti-CD19 Antibodies.

Native Isoelectric Focusing Polyacrylamide Gel Electrophoresis(IEF-PAGE) analysis was performed on 16C4, 16C9, 7E12, 14H5, 15D7, 15D1,14H5-DG, and 3649 anti-CD19 antibodies following standard protocols.Pre-cast ampholine gels (Amersham Biosciences, p1 range 3.5-9.5) wereloaded with 8 μg of purified protein. Protein samples were dialyzed in10 mM Histidine pH 6.0 buffer before loading on the gel. Broad range p1marker standards (Amersham, p1 range 3-10, 8 μL) were used to determinerelative p1 values. Electrophoresis was performed at 1500 V, 50 mA for105 minutes. The gel was fixed for 45 minutes and stained overnight atroom temperature using Simply Blue stain (Invitrogen). Destaining wascarried out with a 25% ethanol, 8% acetic acid solution. Isoelectricpoints were determined using a Bio-Rad GS-800 Densitometer with QuantityOne Imaging Software. The Coomassie stained gel is shown in FIG. 27. Theisoelectric point of the 16C4, 16C9, 7E12, 14H5, 15D7, 15D1, 14H5-DG,and 3649 antibodies is 7.83, 8.04, 7.69, 7.76, 7.61, 7.72, 7.48, and7.75, respectively.

7.11.3. Fc Variant Affinity Matured Anti-CD19 Antibodies

Antibody expression vectors encoding a 16C4 Fc variant antibodycomprising the L234F/L235F/P331S or L234F/L235Y/P331S amino acidsubstitutions (hereinafter referred to as “16C4-235F” or “16C4-235Y”) isgenerated using methods described in US 2004/0132101 and US2005/0054832, both to Lazar et al. Briefly, the antibody expressionvector encoding 16C4 is modified using a site directed mutagenesis kit(e.g., QuickChange (Promega)) by introducing the necessary nucleotideresidue substitutions into the polynucleotide sequence encoding theheavy chain constant region to generate the 16C4-235F or 16C4-235Yantibody expression vector. Purified 16C4 Fc variant antibody isgenerated by transfecting HEK239F cells with the appropriate antibodyexpression vector. Transfected cells are fed at day 3 and 6 and theantibody-containing conditioned medium is harvested at day 9. Antibodyis purified from the conditioned medium using a pre-cast protein Acolumn (GE Healthcare). Antibody is eluted from the column with low pHbuffer, neutralized, and dialyzed against PBS. The concentration of thepurified antibody is calculated from the solution's optical density at280 nm.

Measurement of Equilibrium Binding Constants (K_(D)): The equilibriumbinding constants of all Fey receptors (human FcγRI, FcγRIIA, FcγRIIB,FcγRIIIA-V158, as well as murinc FcγRIIB, FeγRIII and FeγRIV) to 16C4and its Fc variants was measured on a BIAcore 3000 instrument (Uppsala,Sweden). Briefly, all IgGs were immobilized onto separate flow cells oftwo CMS sensor chips using standard amino coupling chemistry asrecommended by the manufacturer. Immobilized IgG levels ranged from 8194to 8725 RUs. Stock solutions of the recombinantly expressedextracellular domains of all FcγRs at either 4000 or 16000 nM wereprepared and then serially diluted down to the desired concentrationsusing the instrument buffer (50 mM HBS buffer containing 0.01 M HEPES,pH 7.4, 0.15 M NaCl, 3 mM EDTA and 0.005% P-20). Duplicate injections ofeach concentration of FcγR were then injected over all of the IgGsurfaces at a flow rate of 5 μL/min. Binding data were collected forapproximately 50 min, followed by a 30 sec. pulse of 5 mM HCl betweeninjections to regenerate the IgG surfaces. Several buffer injectionswere also interspersed throughout the injection series. One of thesebuffer injections was used along with the reference cell data to correctthe raw data sets. After all binding data was collected, individual datasets were averaged for each y concentration, then fit to a 1:1 bindingisotherm from which the equilibrium binding constants, K_(D), werederived. This was carried out using the BIAevaluation software, v. 4.1.K_(D) values (nM) are presented in Table 38.

TABLE 38 Binding affinities (KD, nM) of various human IgGls to human andmouse FcγR. 16C4 16C4-235F 16C4-235Y Human FcγRI 19 1530 8650 HumanFcγRIIA 1280 6360 6980 Human FcγRIIB 14500 6810 17100 Human FcγRIIIA 5744610 5140 (V158) Mouse FcγRIIB 1470 2820 2670 Mouse FcγRIV 329 11100 N/AMouse FcγRIII 6360 10900 9240

7.12. Isolation of Affinity Matured Variants of the 16C4 Anti-CD19Antibody

Affinity matured variants of the 16C4 antibody were identified using theprotocols described herein. The screen consisted of two stages. Thefirst stage focused on the identification of 16C4 variant Fabscomprising single amino acid substitution that resulted in increasedbinding activity to cell surface displayed human CD19 antibodies. 16C4variant Fabs comprising a beneficial single amino acid substitution wereidentified via screening single CDR focused phage display libraries. Thesecond stage of the screen consisted of screening a combinatoriallibrary of Fab clones representing all possible combinations ofbeneficial single amino acid substitutions identified either (i) in thefirst stage of the 16C4 affinity maturation process or (ii) during theaffinity maturation process of the 3649 anti-CD19 antibody.

CDR specific phage display libraries were generated as described above.Libraries were screened by testing a large number of phage clones(approximately 400 clones per library) in single point cell basedbinding assay (Lu et al., J. Immunol. Methods 314:74-79 (2006)).Reagents and disposables were purchased from Meso Scale Discovery;assays were performed following the manufacturer's instructions.Briefly, 5,000 Raji or 300B4 cells/well were plated and incubated for 1hr at RT in 25 μl 1×PBS; wells were blocked with 25 μl 30% FBS for 20min RT; supernatant is discarded; 25 μl of anti-CD19 Ab added into eachwell and incubated for 1 hr at RT; wells are washed 3× with 1×PBS; add25 μl 0.25 μg/ml goat-anti-human Fab′2-MSDTag to each well and incubatefor 1 hr at RT; wash wells 3× with I×PBS; signal is read with 150 μl of1×T Read Buffer. The binding curve of a representative clone comprisinga beneficial amino acid substitution in VH CDR2 is shown in FIG. 32.Beneficial single amino acid substitutions identified from the 16C4 CDRspecific libraries are listed in Table 39.

TABLE 39 Beneficial single amino acid substitutions identified from the16C4 antibody based CDR specific phage display libraries. CloneSubstitution Position VH CDR1 17B7 Ser > Val 32 VH CDR2 64D4 Pro > Leu52A VL CDR2 40A5 Gln > Arg 54 40C10 Gln > Thr 54 43A10 Gln > Ala 54 VLCDR3 2F7 Gln > Ala 89

Combinatorial phage display libraries were prepared as described above.16C4 Fab specific oligonucleotides used for library generation arelisted in Table 40. Individual Fab clones from the combinatorial librarywere tested for binding to 300B4 and Raji cells (Lu et al., J. Immunol.Methods 314:74-79 (2006)). Binding curves of representative Fab clonesare shown in FIG. 33A-B. Fab clones with increased binding activity to300B4 cells, Raji cells, or both were sequenced using standard methods.A summary of amino acid changes found in the CDR sequences of unique Fabclones is presented in FIG. 33C.

TABLE 40 Degenerate nucleotides for combinatorialphage library generation. SEQ Name ID NO. L35 CDR2LGGACCCCGGATCCTSTATTGGATGCCTC 176 p5T/R ATGG L36 CDR2LCCTCGAGGGGACCCCGGAGTATSTATTG 177 p5T/R, p6Y GATGCCTCATGGATG L37 CDR2LGGACCCCGGATCCTGSATTGGATGCCTC 178 p5A/P ATGG L38 CDR2LCCTCGAGGGGACCCCGGAGTATGSATTG 179 p5A/P, p6Y GATGCCTCATGGATG L39 CDR3LCTCCGCCGAACGTGWTTGGAACCTCCTT 180 p3S/T, p8N/I AGWTTGCTGACAGTAATACGL40 CDR3L CGCCGAACGTGAATGGAACCCGCTTAGW 181 p1A/P, p3S/T,TTGCG CACAGTAATACGTTGCAGC 5R L41 CDR3L CGCCGAACGTGAATGGAACCCGCTTAGW 182p3S/T, p5R TTGCTGACAGTAATACGTTG L42 CDR3L CTCCGCCGAACGTGWTTGGAACCTCCTT183 p1A/P, p3S/T, AGWTTGCGCACAGTAATACGTTGC p8N/I H43 CDR1HCCTGGCGGACCCAGTTCATCCAAACGCT 184 p2V ACTGAAGGTGAATCC H44 CDR1HGCCTGGCGGACCCAGTWCATCMAAACGC 185 p2V, p3W/L, TACTGAAGGTGAATC p5N/YH45 CDR1H GAGCCTGGCGGACCCAGAGCATCMAAAC 186 p2V, p3W/L, GCTACTGAAGGTGAATCp5L H46 CDR2H CTTGAACTTCACATWGTAGTTAKTATCT 187 p4L/P, p9T/N,CCATCTCCARGATAAATCCGGCCA p12N/Y H47 CDR2H CTTGAACTTCACATTGTAGTTAKTATCT188 p4L/P, p9T/N CCATCTCCARGATAAATCCGGCCA H48 CDR2HCTTGAACTTCACATWGTAGTTAGTATCT 189 p4L/P, p12N/Y CCATCTCCARGATAAATCCGGCCAH49 CDR3H GTCAAAGTCGCGAACCGTAGKAATAAAT 190 p5P/T CCTGATCTAGC

Six affinity matured 16C4 variant Fab clones with improved bindingactivity to cell surface displayed human CD19 antigen were transformedinto full IgG1 antibodies using methods described herein. The bindingactivity of 3C3, 6F7, 2B11, 6C11, 9G7, and 5C4 affinity maturedanti-CD19 antibodies were characterized in various cell based assays.FIG. 34 presents the results obtained using 300.B4 cells in a cell basedECL assay (Lu et al., J. Immunol. Methods 314:74-79 (2006)). The CD19binding activity of affinity matured 16C4 variant antibodies is higherthan that of the control 16C4 or 3649 antibodies.

Cell surface staining by affinity matured anti-CD19 antibodies. Daudiand Raji cells were immunostained with 3C3, 6C11, and 9G7 anti-CD19antibodies and a RPE conjugated goat anti-human IgG (Fab′)2 fragmentsecondary reagent according to standard protocols. Immunostained cellswere analyzed on a flow cytometer. Median fluorescence intensity (MFI)of stained cells at various primary antibody concentrations is plottedin FIG. 35. Cells stained with the 16C4 anti-CD19 antibody were includedas a reference control. Staining intensity detected with the 3C3 and6C11 affinity matured antibodies on Raji cells at 0.0625-0.25 jug/mlantibody concentration is higher than that of detected with the 16C4control. Raji cell staining intensity of the affinity matured andcontrol antibodies is the same at 0.5-10 μg/ml antibody concentration.Raji cell staining by the 9G7 antibody is similar to the controlantibody at lower (0.0625-0.25 μg/ml) antibody concentrations and weakerthan the control at higher (0.5-10 μg/ml) antibody concentrations.Median FI of 9G7 and 6C11 stained Daudi cells is higher than that of the16C4 stained cells. Median FT of 3C3 stained Daudi cells is higher thanthe control cells at 0.0625 and 0.125 μg/ml antibody concentration; andsubstantially the same as that of the control cells at 0.25-10 μg/mlantibody concentration.

In vitro ADCC activity of affinity matured 16C4 variant anti-CD19antibodies. In vitro ADCC activity of affinity matured anti-CD19antibodies was measured using assays described herein. For example,results obtained with 3C3, 6C11, and 9G7 antibodies using Raji and Dauditarget cells are presented in FIGS. 36 and 37, respectively. The 16C4anti-CD19 antibody was used as a reference control. All three affinitymatured antibody displayed substantially the same ADCC activity as thecontrol over the concentration range tested 90.0001-10 μg/ml).

It is understood by persons of skill in the art that the affinitymatured variants of the 16C4 anti-CD19 antibody may be further modifiedaccording to the protocols described herein. Specifically, the 3C3,6C11, and 9G7 antibodies may be modified to comprise any one of thevariant Fc regions described herein. An afucosylated version of theantibodies may also be prepared. The affinity matured antibodies mayalso be characterized using the assays described herein. Specifically,the ability of the 3C3, 6C11, and 9G7 antibodies to mediate ADCC, to invivo deplete B cells, to reduce the size of tumor xenografts, to inhibitanti-IgM/CpG stimulated B cell proliferation may be tested following theprotocols described herein.

7.13. Anti-CD19 Antibody Mediated Inhibition of B Cell Proliferation7.13.1. Anti-CD19 Antibody Treatment Induced CD19 Phosphorylation

Ten million cells were incubated for 15 minutes in the presence of 5μg/ml 3649, 3649-3M, 3649-aFuc, 3649-TM or 16C4 anti-CD19 antibody.Control cells treated with the R347 antibody of irrelevant specificityas well as control cells without antibody treatment were included in theexperiment as negative controls. Following incubation, cell lysates wereprepared and subjected to immunoprecipitation according to standardprotocols. Immunoprecipitated material and input cell lysate wereseparated on a Laemmli SDS-PAGE, transferred to solid support(Nitrocellulose Invitrogen Cat# LC2001) and subjected to Westernblotting. Immunoprecipitation was performed with 2 μg HB12B anti-CD19antibody following standard protocols. Total CD19 protein andphosphorylated CD19 levels were detected in the immunoprecipitatedmaterial by Western blotting using the (1:1000) anti-CD19 (CellSignaling Technology #3574) or (1:250) anti-phospho tyrosine (PY20)(Santa Cruz Biotechnology #sc-508 HRP) antibody, respectively.Phosphorylated Erk1/2 protein and total Erk1/2 protein levels in theinput cell lysate were detected by Western blotting using the (1:2000)anti-phospho Erk1/2 (Cell Signaling Technology #9106S) and (1:1000)anti-Erk1/2 (Cell Signaling Technology #9102) antibody, respectively.

FIG. 39A shows the results of HB12B immunoprecipitation followed byWestern blotting. In addition to the immunoprecipitated samples from thevarious experimental (“3649”, “3649-3M”, “3649-aFuc”, “3649-TM” or“16C4”) and control cell lysates (“nil” and “R347”), the membrane alsocontained an HB12B antibody only control lane (“Ab alone”). Total CD19protein levels were substantially identical in all immunoprecipitatedsamples. Phosphorylated CD19 levels were significantly higher in thesamples immunoprecipitated from cell lysates prepared from the anti-CD19treated cells than in the control samples. FIG. 39B shows the Westernblot results on total cell lysates. Total Erk1/2 protein levels weresubstantially identical in all cell lysates. Phosphorylated Erk1/2levels were significantly higher in the cell lysates prepared from theanti-CD19 treated cells than in the control samples.

7.13.2. Anti-CD19 Treatment does not Inhibit Anti-IgM Mediated Erk1/2Activation.

One million cells were stimulated with 5 μg/ml anti-IgM antibody or PMA(50 ng/ml)/ionomycin (1 μM) for five or ten minutes in the presence ofeither 10 μg/ml 3648-3M anti-CD19 antibody or 10 μg/m R347 controlantibody. Cells with only 3649 or R347 treatment were included ascontrols. Cells were harvested and lysed at the end of the incubationperiod. Total cell lysate was separated on a Laemmli SDS-PAGE,transferred to a nitrocellulose support membrane and subjected toWestern blotting following standard protocols. Western blotting usinganti-phospho Erk1/2 antibody and anti-Erk1/2 antibody was performed todetect phosphorylated Erk1/2 and total Erk1/2 levels, respectively, inthe cell lysate. Results are shown in FIG. 39C-D.

Total Erk1/2 levels were substantially identical in all cell lysates.Baseline phosphorylated Erk1/2 level in 3649 antibody only treated cellswas higher than that of R347 only treated cells. Anti-IgM orPMA/ionomycin stimulation increased phosphorylated Erk1/2 levels abovethe baseline in both 3649 or R347 treated cells. Erk1/2 phosphorylationlevel was significantly higher following PMA/ionomycin stimulation thanafter stimulation with anti-IgM antibody.

7.13.3. Anti-CD19 Antibody Treatment Inhibits Anti-IgM/CD40 Induced BCell Proliferation.

Peripheral B cells are purified from 200 mls of blood using a B cellisolation kit (Miltenyi Biotec #130-091-151). 100,000 cells are seededin a 96-well U-bottomed plate (100 ul of 1×10⁶ cell/ml). Next, theappropriate concentration of antibody is added to the cells in a 50 ulvolume. The plate is returned to the incubator for 15 minutes. Then, thestimulus is added to the cells in a 50 μl volume. The final volume ofcell/antibody/stimulus mixture is 200 ul. Cells are incubated for threedays. Cell numbers are read on day three using the CellTiter-Glo®Luminescent Cell Viability Assay (Promega). Experiments on immortalizedcell lines were seeded with 10,000 cells per well.

The effect of anti-CD19 antibody treatment on anti-IgM/CD40 induced Bcell proliferation is shown in FIG. 40. B cells were plated in thepresence of 10 i.tg/ml 3649, 3649-TM, and 3649-3M anti-CD19 antibody. 15minutes later cells B cells were stimulated with anti-IgM (5 μg/ml)alone, anti-IgM (5 μg/ml)/CD40 (1 μg/ml), or CpG (1 μg/ml) alone. B cellstimulation was allowed to proceed for three days. Viable cell numberswere measured at the end of the experiment using the CellTiter-Glo®Luminescent Cell Viability Assay (Promega). Cells treated with the R347antibody of irrelevant specificity were included as control. Viable cellnumbers were increased by anti-IgM/CD40 or CpG stimulation, but not byIgM alone stimulation. Anti-IgM/CD40 induced cell proliferation wassignificantly inhibited by anti-CD19 antibody treatment. Level ofinhibition (40%) was identical for all anti-CD19 antibodies tested. CpGinduced cell proliferation was unaffected by anti-CD19 antibodytreatment.

7.13.4. Anti-CD19 Antibody Treatment Inhibits Anti-IgM/CpG Induced BCell Proliferation.

Cell proliferation in response to various stimuli was assessed using theCFSE assay. Briefly, purified B cells are re-suspended in phosphatebuffered saline (PBS) at approximately 10 million cells per milliliter.To that an equal volume of 1 uM CFSE in PBS is added. The finalconcentration of CFSE is 0.5 uM and the cells are at 5 million permilliliter. The suspension is kept in the dark for 10 minutes. An equalvolume of Fetal Calf Serum (FCS) is added to the mixture to quenchextracellular CFSE. Cells are washed and diluted in media. 100,000 CFSElabeled cells are seeded in a 96-well U-bottomed plate (100 ul of 1×10⁶cell/ml). Next, the appropriate concentration of antibody is added tothe cells in a 50 ul volume. The plate is returned to the incubator for15 minutes. Next, the stimulus is added to the cells in a 50 μl volume.The final volume of cell/antibody/stimulus mixture is 200 ul. Cells areincubated for four days. At the end of incubation, cells are washed,stained with 7-amino actinomycin D (7-AAD) (BD Bioscience), and analyzedon a flow cytometer. CFSE signal of live cells is detected. CFSE signaldecrease in the CFSE profile of a cell population is indicative of celldivision. The extent of CFSE signal decrease correlates with cellproliferation levels.

Purified peripheral B cell were stimulated with anti-IgM (1 μg/ml)/CpG(2 μg/ml) or CpG (2 μg/ml) alone for four days. Cell proliferation isassessed using the CFSE assay. FIG. 41A shows the CFSE profile ofstimulated and unstimulated control cells. The CFSE signal of IgM (1μg/ml)/CpG (2 μg/ml) stimulated cells is significantly lower than thatof the control cells indicating that IgM (1 μg/ml)/CpG (2 μg/ml)stimulation resulted in extensive cell proliferation. The CFSE profileof CpG only stimulated cells indicates only limited cell proliferation.

16C4 anti-CD19 antibody inhibits anti-IgM/CpG induced B cellproliferation. Purified peripheral B cells were stimulated with anti-IgM(1 μg/ml)/CpG (2 μg/ml) in the presence of 5 μg/ml of R347 controlantibody or 5 μg/ml 16C4 anti-CD19 antibody for four days. Cellproliferation is assessed using the CFSE assay. FIG. 41B shows the CFSEprofile of B cells stimulated with IgM (1 μg/ml)/CpG (2 μg/ml) in thepresence of R347 or 16C4 antibody. The CFSE profile of B cellsstimulated with anti-IgM (1 μg/ml)/CpG (2 μg/ml) in the presence of R347antibody is indicative of extensive cell proliferation. The CFSE profileof B cells stimulated with anti-IgM (1 μtg/ml)/CpG (2 μg/ml) in thepresence of 16C4 anti-CD19 antibody is indicative of less extensive cellproliferation than that of seen in the control cells.

7.13.5. Fc Variant Anti-CD19 Antibodies Display Altered InhibitoryProperties.

Purified peripheral B cells were stimulated for four days with anti-IgM(1 μg/ml)/CpG (2 μg/ml) in the presence of 5 μg/ml of R347 controlantibody, 3649-3M Fc variant anti-CD19 antibody or 3649-TM Fc variantanti-CD19 antibody. Cell proliferation is assessed using the CFSE assay.FIG. 42A shows the CFSE profile of B cells stimulated with anti-IgM (1μg/ml)/CpG (2 μ/ml) in the presence of R347 control antibody. The CFSEprofile shows that 23.5+26.5+23.2=73.2% of the B cells underwent atleast one round of cell division. FIGS. 42B and C shows the CFSE profileof B cells stimulated with anti-IgM (1 μg/ml)/CpG (2 μg/ml) in thepresence of 3649-TM and 3649-3M Fc variant anti-CD19 antibody,respectively. The CFSE profiles show that 44.8% and 30.3% of B cellsstimulated in the presence of 3649-TM and 3649-3M Fc variant anti-CD19antibody, respectively, underwent at least one round of cell divisionduring the four day long incubation period. Closer inspection of allthree CFSE profiles reveals that not only the number of cells undergoingat least one division, but also the number of cells undergoing more thanone divisions drops with highest and lowest cell proliferation seen inthe presence of R347 and 3649-3M, respectively. Treatment with 3649-TMinhibits cell proliferation less effectively than treatment with3649-3M.

Purified B cells were stimulated for four days with anti-IgM (5μg/ml)/CpG (1 μg/ml) in the presence of 5 μg/ml R347, R347-3M F cvariant, 3649, 3649-3M Fc variant, or 3649-TM Fc variant antibodies.Cell proliferation is assessed using the CFSE assay. CFSE profile of Bcells stimulated in the presence of R347 is included in all panels as areference standard. CFSE profiles of B cells stimulated in the presenceof R347-3M F c variant, 3649, 3649-3M Fc variant, and 3649-TM Fc variantantibodies is shown in panels A-D. Cell proliferation in the presence ofR347-3M Fc variant is same as the one seen in the presence of the R347reference standard. Cell proliferation is inhibited by all threeanti-CD19 antibodies. The wild type 3649 and 3649-TM Fc variantantibodies inhibited cell proliferation to the same extent. 3649-3M wasmore effective at inhibiting B cell proliferation than either the 3649or 3649-TM antibodies.

Inhibitory synergism of anti-CD19 and anti-Fcgamma receptor IIB (FcγRIIbor CD32b) antibodies: The following experimental design will furthertest whether there is a synergistic interaction between the anti-CD32band anti-CD19 antibody mediated inhibitions of B cell proliferation.Purified B cells will be stimulated for four days with anti-IgM (2μg/ml)/CpG (2 μg/ml) in the presence of (i) an anti-CD32b antibody(e.g., AT10), (ii) an anti-CD19 antibody or (iii) both anti-CD32b andCD19 antibodies. Cell proliferation will be assessed using the CFSEassay. Synergism between anti-CD32b and anti-CD19 antibody mediatedinhibition of B cell proliferation is expected to lead to lower B cellproliferation in the presence of both antibodies than the cellproliferation seen in the presence of either antibody alone.

7.13.6. Anti-CD19 Antibodies are Efficiently Internalized.

Antibody internalization assay: Cells are incubated with 5 i.ig/ml AlexaFlour 488-labeled antibodies at 37° C. for up to 60 minutes. An aliquotof cells are removed at 10 minute intervals, washed, divided into twoparts, and placed on ice. One half aliquot is left untreated on ice. Thesecond half aliquot is treated on ice for 45 seconds with a low pH (2.0)PBS solution containing 0.03M sucrose and 10% FCS to strip all cellsurface bound antibody molecules. Both acid treated and untreatedsamples are washed, fixed with 4% paraformaldehyde, and analyzed on aflow cytometer. % internalized antibody is calculated as the ratio ofthe fluorescence signal of acid washed cells (internal signal only) andthat of untreated cells (total signal from cell surface and internalcompartments).

FIG. 44 shows the internalization of HB12B, 3649, and 16C4 antibodies byRaji cells over a 60 minute time period. Internalization curves showthat anti-CD19 uptake reaches a maximum around 20-30 minutes. Maximuminternalization is about 50% for the HB12B and 3649 antibodies and ˜30%for the 16C4 antibody.

7.13.7. Loss of CD19 from the Cell Surface Following 24 Hrs of Anti-CD19Antibody Treatment.

Cells are incubated in the presence of an anti-CD19 antibody for 24hours. A control cell population is incubated in the presence of theR347 antibody of irrelevant specificity. Following incubation, cells areharvested, washed and incubated on ice for 10 minutes in a stainingsolution comprising 5 μg/ml anti-CD19 antibody for 10 minutes. Surfacebound anti-CD19 antibody is detected by staining the cells for 10minutes with a PE conjugated goat anti-human IgG secondary antibody.Immunostained cells are fixed in 4% paraformaldehyde and analyzed on aflow cytometer. CD19 surface loss is calculated by comparing the meanfluorescence intensity (MFI) of anti-CD19 antibody treated cells to theMFI of immunostained R347 treated cells (0% surface loss) and secondaryantibody only stained R347 treated cells (100% surface loss).

FIG. 45 displays the CD19 surface loss on Raji cells and primary B cellsfollowing incubation for 24 hours in the presence of 5 μg/ml of 3649,3649-3M, 3649-TM, 3648-aFuc, and 16C4 anti-CD19 antibodies. A 55-70% and65-90% loss of CD19 surface expression is detected in Raji cells andprimary B cells, respectively, following treatment with anti-CD19antibodies.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties for all purposes. Thedisclosure of US Provisional Application Nos. 60/842,935 filed Sep. 8,2006, 60/866,917, filed Nov. 22, 2006, 60/911,397, filed Apr. 12, 2007,60/915,309, filed May 1, 2007, and 60/939,429, filed May 22, 2007 areincorporated by reference herein in their entirety for all purposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. An isolated purified chimeric, humanized or humanmonoclonal antibody or fragment thereof comprising a VH comprising theamino acid sequence of SEQ ID NO.:237 or a VL comprising the amino acidsequence of SEQ ID NO.:238, wherein said antibody binds a human CD19antigen.
 2. The antibody of claim 1, wherein said antibody comprises aVH comprising the amino acid sequence of SEQ ID NO.:237 and a VLcomprising the amino acid sequence of SEQ ID NO.:238.
 3. The antibody ofclaim 1, wherein said VH comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO.: 34, 44, 102, 103, 104, 105, 106,107, 108, 109, 191, 192, and 236; and wherein said VK comprises an aminoacid sequence selected from the group consisting of: SEQ ID NO.: 52, 62,68, 70, 74, 76, 78, 80, 110, 111, 193, 194, 195, 196, 917, 198, 199,200, 201, 202, 203, 204, 205, 206, and
 207. 4. The antibody of claim 2,wherein said VH comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NO.: 34, 44, 102, 103, 104, 105, 106, 107, 108,109, 191, 192, and 236; and wherein said VK comprises an amino acidsequence selected from the group consisting of: SEQ ID NO.: 52, 62, 68,70, 74, 76, 78, 80, 110, 111, 193, 194, 195, 196, 917, 198, 199, 200,201, 202, 203, 204, 205, 206, and
 207. 5. The antibody of claim 4,wherein said antibody is selected from the group consisting of: a) anantibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:34 and VL comprising the amino acid sequence of SEQ ID NO.: 68, b)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:102 and VL comprising the amino acid sequence of SEQ ID NO.:110, c)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:103 and VL comprising the amino acid sequence of SEQ ID NO.:111, d)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:103 and VL comprising the amino acid sequence of SEQ ID NO.:113, e)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:104 and VL comprising the amino acid sequence of SEQ ID NO.:112, f)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:105 and VL comprising the amino acid sequence of SEQ ID NO.:111, g)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:106 and VL comprising the amino acid sequence of SEQ ID NO.:111, h)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:107 and VL comprising the amino acid sequence of SEQ ID NO.:111, i)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:108 and VL comprising the amino acid sequence of SEQ ID NO.:111, j)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:109 and VL comprising the amino acid sequence of SEQ ID NO.:111, k)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:111, l)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:193, m)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:194, n)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:195, o)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:192 and VL comprising the amino acid sequence of SEQ ID NO.:196, p)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:197, q)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:198, r)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:106 and VL comprising the amino acid sequence of SEQ ID NO.:199, s)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:106 and VL comprising the amino acid sequence of SEQ ID NO.:200, t)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:201, u)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:202, v)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:106 and VL comprising the amino acid sequence of SEQ ID NO.:203, w)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:106 and VL comprising the amino acid sequence of SEQ ID NO.:198, x)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:192 and VL comprising the amino acid sequence of SEQ ID NO.:204, y)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:205, z)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:206, aa)an antibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:197, andbb) an antibody comprising a VH comprising the amino acid sequence ofSEQ ID NO.:191 and VL comprising the amino acid sequence of SEQ IDNO.:207.
 6. The antibody of claim 4, wherein said antibody possesses afunctional activity selected from the group consisting of: enhanced ADCCactivity, induction of B cell apoptosis, inhibition of B cellproliferation, and in vivo B cell depletion.
 7. The antibody of claim 4,wherein said antibody has complex N-glycoside-linked sugar chains boundto the Fc region in which fucose is not bound to N-acetylglucosamine inthe reducing end in the sugar chain.
 8. The antibody of claim 7, whereinsaid antibody efficiently mediates in vitro ADCC activity againstKarpas-422, Karpas-1106P, and DB cell lines but not against Granta-519cell line.
 9. The antibody of claim 7, wherein said antibody is capableof depleting B cells in an animal model, wherein said B cells areselected from a group consisting of: circulating B cells, blood B cells,splenic B cells, marginal zone B cells, follicular B cells, peritoneal Bcells, and/or bone marrow B cells.
 10. The antibody of claim 9, whereinsaid depletion reduces B cell levels by at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95% seven days afteradministration of 2.5 mg/kg dose of said antibody.
 11. The antibody ofclaim 4, wherein said antibody is an Fc variant, wherein said variant Fchas an altered affinity for one or more Fc ligand selected from thegroup consisting of: Clq, FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIV.12. The antibody of claim 11, wherein said Fc variant has an affinityfor the Fc receptor FcγRIIIA that is at least about 5 fold lower thanthat of a comparable molecule, and wherein said Fc variant has anaffinity for the Fc receptor FcγRI1B that is within about 2 fold of thatof a comparable molecule.
 13. The antibody of claim 12, wherein said Fcvariant comprises mutations that result in enhanced ADCC activity. 14.An isolated nucleic acid encoding a polypeptide comprising an amino acidsequence selected from the group consisting of: SEQ ID NO: 34, 44, 102,103, 104, 105, 106, 107, 108, 109, 191, 192, 236, 52, 62, 68, 70, 74,76, 78, 80, 110, 111, 193, 194, 195, 196, 917, 198, 199, 200, 201, 202,203, 204, 205, 206, and
 207. 15. An isolated cell expressing theantibody of claim
 4. 16. A pharmaceutical composition comprising theantibody of claim 4 in a pharmaceutically-acceptable carrier.
 17. Amethod of treating a B cell disease or disorder in a human comprising:administering to a human in need thereof a therapeutically-effectiveamount of an antibody, a) wherein said antibody is a chimeric, humanizedor human monoclonal antibody or fragment thereof, b) wherein saidantibody comprises a VH or a VL wherein said VH comprises an amino acidsequence selected from the group consisting of: SEQ ID NO.: 34, 44, 102,103, 104, 105, 106, 107, 108, 109, 191, 192, and 236, c) wherein said VLcomprises an amino acid sequence selected from the group consisting of:SEQ ID NO.: 52, 62, 68, 70, 74, 76, 78, 80, 110, 111, 193, 194, 195,196, 917, 198, 199, 200, 201, 202, 203, 204, 205, 206, and 207, d)wherein said antibody binds a human CD19 antigen, and e) wherein saiddisease or disorder is selected from a group consisting of: B cellmalignancy, autoimmune disease, autoimmune disorder, humoral rejectionin a human transplant patient, graft-versus-host disease (GVHD) andpost-transplantation lymphoproliferative disorder in human transplantrecipient.
 18. The method of claim 17, wherein said antibody is selectedfrom the group consisting of: a) an antibody comprising a VH comprisingthe amino acid sequence of SEQ ID NO.:34 and VL comprising the aminoacid sequence of SEQ ID NO.: 68, b) an antibody comprising a VHcomprising the amino acid sequence of SEQ ID NO.:102 and VL comprisingthe amino acid sequence of SEQ ID NO.:110, c) an antibody comprising aVH comprising the amino acid sequence of SEQ ID NO.:103 and VLcomprising the amino acid sequence of SEQ ID NO.:111, d) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:103 andVL comprising the amino acid sequence of SEQ ID NO.:113, e) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:104 andVL comprising the amino acid sequence of SEQ ID NO.:112, f) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:105 andVL comprising the amino acid sequence of SEQ ID NO.:111, g) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:106 andVL comprising the amino acid sequence of SEQ ID NO.:111, h) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:107 andVL comprising the amino acid sequence of SEQ TD NO.:111, i) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:108 andVL comprising the amino acid sequence of SEQ ID NO.:111, j) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:109 andVL comprising the amino acid sequence of SEQ ID NO.:111, k) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:111, l) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:193, m) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:194, n) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:195, o) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:192 andVL comprising the amino acid sequence of SEQ ID NO.:196, p) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:197, q) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:198, r) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:106 andVL comprising the amino acid sequence of SEQ ID NO.:199, s) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:106 andVL comprising the amino acid sequence of SEQ ID NO.:200, t) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:201, u) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:202, v) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:106 andVL comprising the amino acid sequence of SEQ ID NO.:203, w) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:106 andVL comprising the amino acid sequence of SEQ ID NO.:198, x) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:192 andVL comprising the amino acid sequence of SEQ TD NO.:204, y) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:205, z) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:206, aa) an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO.:191 andVL comprising the amino acid sequence of SEQ ID NO.:197, and bb) anantibody comprising a VH comprising the amino acid sequence of SEQ IDNO.:191 and VL comprising the amino acid sequence of SEQ ID NO.:207. 19.The method of claimed 17, wherein said method comprises the depletion ofB cells selected from the group consisting of: circulating B cells,blood B cells, splenic B cells, marginal zone B cells, follicular Bcells, peritoneal B cells, and/or bone marrow B cells.
 20. The method ofclaimed 17, wherein said method comprises the depletion of B cellsselected from the group consisting of: progenitor B cells, early pro-Bcells, late pro-B cells, large-pre-B cells, small pre-B cells, immatureB cells, mature B cells, antigen stimulated B cells, and/or plasmacells.
 21. The method of claim 17, wherein said antibody has an enhancedADCC activity.
 22. The method of claim 21, wherein said depletionreduces B cell levels by at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, orabout 100%.
 23. The method of claim 21, wherein said depletion persistfor a time period selected from the group consisting of: at least 1week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3months, at least 4 months, at least 5 months, at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months or at least 12 months.